Chfr is required for tumor suppression and Aurora A regulation.

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.

Detection of protein-RNA complexes in Xenopus oocytes.

There is a remarkable variety of mechanisms for controlling post-transcriptional gene expression that is achieved through the formation of ribonucleoprotein (RNP) complexes on specific cis-acting regions of mRNA. These complexes regulate splicing, nuclear and cytoplasmic polyadenylation, stability, localization, and translation. Thus, it is important to be able to detect the association of specific proteins with specific RNAs within the context of these RNP complexes. We describe a method to test for protein-RNA complexes in Xenopus oocytes. The procedure combines immunoprecipitation with reverse transcription-PCR (RT-PCR) and does not entail chemical or photo crosslinking. Microinjected mRNA is efficiently translated in Xenopus oocytes; thus, in cases where primary antibody is not available, an epitope-tagged version of the protein can be expressed for utilization in this procedure. The inclusion of control mRNAs has provided no evidence of nonspecific protein reassociation to RNA during or subsequent to cell lysis. The method has been used to document the association of certain trans-acting factors specifically with localized mRNAs in Xenopus oocytes.

RCS1, a substrate of APC/C, controls the metaphase to anaphase transition.

The anaphase-promoting complex/cyclosome (APC/C) controls the onset of anaphase by targeting securin for destruction. We report here the identification and characterization of a substrate of APC/C, RCS1, as a mitotic regulator that controls the metaphase-to-anaphase transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G(1). Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and degraded during mitotic exit in a Cdh1-dependent manner in vivo. APC/C recognizes a unique D-box at the N terminus of RCS1, as mutations of this D-box abolished ubiquitination in vitro and stabilized the mutant protein in vivo. RCS1 controls the timing of the anaphase onset, because the loss of RCS1 resulted in a faster progression from the metaphase to anaphase and accelerated degradation of securin and cyclin B. Biochemically, mitotic RCS1 associates with the NuRD chromatin-remodeling complex, and this RCS1 complex is likely involved in regulating gene expression or chromatin structure, which in turn may control anaphase onset. Our study uncovers a complex regulatory network for the metaphase-to-anaphase transition.

Cep55, a microtubule-bundling protein, associates with centralspindlin to control the midbody integrity and cell abscission during cytokinesis.

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.

Monoclonal antibodies to fibroblast growth factor receptor 2 effectively inhibit growth of gastric tumor xenografts.

PURPOSE: Overexpression of fibroblast growth factor receptor 2 (FGFR2) may be a causative factor of a number of human tumors, especially gastric tumors of the poorly differentiated type. We investigated whether monoclonal antibodies (mAbs) directed against FGFR2 can inhibit the growth of tumors in xenograft models. EXPERIMENTAL DESIGN: We generated and characterized 3 mAbs that recognize different epitopes on FGFR2: GAL-FR21, GAL-FR22, and GAL-FR23. The ability of the mAbs to recognize the FGFR2IIIb and FGFR2IIIc isoforms of FGFR2 was determined, as was their ability to block binding of FGF ligands to FGFR2. The capability of the mAbs to inhibit FGF-induced FGFR2 phosphorylation and to downmodulate FGFR2 expression was also investigated. Finally, the ability of the anti-FGFR2 mAbs to inhibit tumor growth was determined by establishing xenografts of SNU-16 and OCUM-2M human gastric tumor cell lines in nude mice, treating with each mAb (0.5-5 mg/kg intraperitoneally twice weekly) and monitoring tumor size. RESULTS: Of the 3 mAbs, GAL-FR21 binds only the FGFR2IIIb isoform, whereas GAL-FR22 and GAL-FR23 bind to both the FGFR2IIIb and FGFR2IIIc forms, with binding regions respectively in the D3, D2-D3, and D1 domains of FGFR2. GAL-FR21 and GAL-FR22 blocked the binding of FGF2, FGF7 and FGF10 to FGFR2IIIb. GAL-FR21 inhibited FGF2 and FGF7 induced phosphorylation of FGFR2, and both mAbs downmodulated FGFR2 expression on SNU-16 cells. These mAbs effectively inhibited growth of established SNU-16 and OCUM-2M xenografts in mice. CONCLUSIONS: Anti-FGFR2 mAbs GAL-FR21 and GAL-FR22 have potential for the treatment of gastric and other tumors.

Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis.

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.

MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis.

Initiation of cytokinesis requires the establishment of the cleavage plane, the assembly of the contractile ring, and the ingression of the cleavage furrow. MgcRacGAP, a GTPase-activating protein for RhoA, is required for cytokinesis, but the mechanism of its action remains unknown. We report here that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through the RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNA interference (RNAi) failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase-activating protein, associates during cytokinesis with ECT2, a guanine nucleotide exchange factor for RhoA, and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow.

A homolog of FBP2/KSRP binds to localized mRNAs in Xenopus oocytes.

A Xenopus oocyte expression library was screened for proteins that bind to the 340-nucleotide localization element of Vg1 mRNA. Four different isolates encoded a Xenopus homolog of the human transcription factor, FUSE-binding protein 2 (FBP2). This protein has been independently identified as the splicing regulatory factor KSRP. The only significant difference between the Xenopus protein, designated VgRBP71, and KSRP is the absence of a 58 amino acid segment near the N-terminal of the former. In vivo binding assays show that VgRBP71 is associated with mRNAs localized to either the vegetal or animal hemispheres, but was not found with control mRNAs. Unlike other factors that bind to the localization element of Vg1 mRNA, VgRBP71 does not accumulate at the vegetal cortex with the mRNA; rather, it is present in the nucleus and throughout the cytoplasm at all stages of oogenesis. Cytoplasmic VgRBP71 appears to be most concentrated at the cell cortex. VgRBP71 interacts with Prrp, another protein that binds to the Vg1 localization element; this association does not require the presence of Vg1 mRNA.