Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3, Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex. This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. We generated mice with a null mutation in the Srg3 locus to examine its function in vivo. Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter. An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture. Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly. Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation. Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

The mammalian SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin remodeling complex that consists of nine or more components. SRG3, a murine homologue of yeast SWI3, Drosophila MOIRA, and human BAF155, is a core component of the murine SWI/SNF complex required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. Here we report that SRG3 interacts directly with other components of the mammalian SWI/SNF complex such as SNF5, BRG1, and BAF60a. The SWIRM domain and the SANT domain were required for SRG3-SNF5 and SRG3-BRG1 interactions, respectively. In addition, SRG3 stabilized SNF5, BRG1, and BAF60a by attenuating their proteasomal degradation, suggesting its general role in the stabilization of the SWI/SNF complex. Such a stabilization effect of SRG3 was not only observed in the in vitro cell system, but also in cells isolated from SRG3 transgenic mice or knock-out mice haploinsufficient for the Srg3 gene. Taken together, these results suggest the critical role of SRG3 in the post-transcriptional stabilization of the major components of the SWI/SNF complex.The mammalian SWI/SNF complexes are evolutionarily conserved ATP-dependent chromatin remodeling complexes, which use the energy of ATP hydrolysis to mobilize nucleosomes and remodel chromatin structure (1, 2). These complexes play important roles in transcriptional regulation, thereby controlling diverse cellular processes including proliferation, differentiation, cell death, and tumorigenesis (3-6). The mammalian SWI/SNF complexes are multisubunit complexes that consist of invariant core components and variable components (7). The subunit diversity of mammalian SWI/ SNF complexes suggests that different complexes might have tissue-specific roles during development (8). The core components of the mammalian SWI/SNF complexes are BRG1 or hBRM, SNF5/INI1/BAF47, BAF155/SRG3, and BAF170 (9). BRG1 and BRM are DNA-dependent ATPase homologous to yeast SWI2/SNF2. Biochemical experiments have shown that although BRG1 or BRM alone can remodel nucleosomal arrays, the addition of other core components (BAF155, BAF170, and SNF5) to BRG1 stimulates the remodeling activity of BRG1 at a rate that is comparable with the entire complex in vitro (10).Human SNF5 was initially identified by the yeast two-hybrid system through its interaction with human immunodeficiency virus type 1 integrase (11). It was shown that human SNF5 interacts with c-Myc, thereby enhancing c-Myc-mediated transactivation by recruiting the SWI/SNF complex to the E-box (12). Furthermore, human SNF5 is known as a tumor suppressor in atypical teratoid and malignant rhabdoid tumors and the majority of these tumors have deletion or point mutations in SNF5 leading to disruption of normal function of SNF5 (13,14).Srg3 (Swi3-related gene), a murine homologue of yeast Swi3, Drosophila Moira, and human Baf155, was initially isolated as a gene highly expressed in the thymus but at a low level in the peripheral lymphoid organ (15). It...

The functional activity of integrins is dynamically regulated by T cell receptor stimulation and by protein kinase C (PKC). We report a novel function for the PKC effector protein kinase D1 (PKD1) in integrin activation. Constitutively active and kinase-inactive PKD1 mutants lacking the PKD1 pleckstrin homology (PH) domain block phorbol ester- and TCR-mediated activation and clustering of beta1 integrins. The PH domain of PKD1 mediates the association of PKD1 with the GTPase Rap1 and is central to Rap1 activation and membrane translocation in T cells. Furthermore, PKD1 and Rap1 associate with beta1 integrins in a manner that is dependent on the carboxy-terminal end of the beta1 integrin subunit cytoplasmic domain. beta1 integrin expression is required for Rap1 activation and membrane localization of the PKD1-Rap1 complex. Therefore, PKD1 promotes integrin activation in T cells by regulating Rap1 activation and membrane translocation via interactions with the beta1 integrin subunit cytoplasmic domain.

The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits. The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells. Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C. Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer. Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits. Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues. The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis. Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP). Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.

A complementary DNA (cDNA) clone that encodes inositol 1,4,5-trisphosphate 3-kinase was isolated from a rat brain cDNA expression library with the use of monoclonal antibodies. This clone had an open reading frame that would direct the synthesis of a protein consisting of 449 amino acids and with a molecular mass of 49,853 daltons. The putative protein revealed a potential calmodulin-binding site and six regions with amino acid compositions (PEST regions) common to proteins that are susceptible to calpain. Expression of the cDNA in COS cells resulted in an approximately 150-fold increase in inositol 1,4,5-trisphosphate 3-kinase activity of these cells.

F emale breast cancer is a major public health problem, with more than 1 000 000 cases occurring worldwide annually. (1) Despite major advances that have been made in understanding the biological and clinical nature of the disease, the therapeutic problem persists. Therefore, identification of novel breast cancer biomarkers could be utilized as possible therapeutic targets or prognostic predictors that would contribute to the advancement of breast cancer treatment.Metastasis associated antigen 1 (MTA1) was originally identified by differential screening of a cDNA library from highly metastatic and non-metastatic rat mammary adenocarcinoma cell lines. (2)(3)(4) The expression level is estimated to be four-fold higher in the highly metastatic cell line MTLn3 than in the non-metastatic cell line MTC4. (2) The mRNA expression level of the human homolog of this gene is approximately four-times higher in a metastatic cell line (MDA-MB-231) than in a non-invasive and non-metastatic cell line (MDA-MB-468) in nude mice. (2) MTA1 is expressed physiologically at low levels in all normal tissue, except the testis. (2) Several studies have identified various roles for MTA1 in normal mammary gland development and human breast cancer progression, including cell proliferation and invasiveness. A study with MTA1 transgenic mice reveals that MTA1 dysregulation in mammary epithelium causes increased cell proliferation, hyper-branched ductal structure formation and precocious development, and results in the development of hyperplastic nodules and mammary gland tumors in virgin mice. (5) The growth of human MDA-MB-231 breast cancer cells is inhibited after treatment with MTA1 antisense phosphorothioate oligonucleotides. (6) MTA1 overexpression in non-invasive breast cancer MCF-7 cells yields larger colony formation in soft agar, augmented colony formation, as well as increased invasiveness in a Boyden chamber assay. (7) Thus, MTA1 may play an important role in tumorigenesis and tumor aggressiveness. However, the role of MTA1 in surgically resected breast cancer tissue with its clinicopathological parameters has not been investigated to date. Therefore, we analyzed the relationship between MTA1 expression and variable clinicopathological features.Tumor microenvironment is known to play an active role in tumor progression through adhesion molecules, angiogenesis and stromal host cells. (8)(9)(10) Previous experimental studies highlight the role of angiogenesis in tumor progression, such as tumor growth and blood-borne metastasis. (11,12) In breast cancer, the overall results of reported studies suggest that human breast cancer is an angiogenic-dependent tumor, and anti-angiogenic therapy may be beneficial for breast cancer patients. (13,14) Therefore, we analyzed the possible role of MTA1 in tumor angiogenesis of breast cancer by counting intratumoral microvessels, which is widely used to estimate tumor angiogenesis. (15) In the present study, we report a statistically significant correlation between MTA1 gene expression and increased...

To understand the tumor-suppressing mechanism of the SWI/SNF chromatin remodeling complex, we investigated its molecular relationship with p53. Using the pREP4-luc episomal reporter, we first demonstrated that p53 utilizes the chromatin remodeling activity of the SWI/SNF complex to initiate transcription from the chromatin-structured promoter. Among the components of the SWI/SNF complex, we identified BAF60a as a mediator of the interaction with p53 by the yeast two-hybrid assay. p53 directly interacted only with BAF60a, but not with other components of the SWI/SNF complex, such as BRG1, SRG3, SNF5, or BAF57. We found out that multiple residues at the amino acid 108 -150 region of BAF60a were involved in the interaction with the tetramerization domain of p53. The N-terminal fragment of BAF60a containing the p53-interacting region as well as small interfering RNA for baf60a inhibited the SWI/SNF complex-mediated transcriptional activity of p53. The uncoupling of p53 with the SWI/SNF complex resulted in the repression of both p53-dependent apoptosis and cell cycle arrest by the regulation of target genes. These results suggest that the SWI/SNF chromatin remodeling complex is involved in the suppression of tumors by the interaction with p53.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)-positive and -negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50-200 lM), independently of its ER status, in a time-and concentration-dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK-3b and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt-mediated canonical Wnt signaling pathway. ER expression was downregulated in ER-positive cells, while c-ErbB2 expression and its activity were suppressed in ER-negative SK-BR-3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA-MB-231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ERpositive and -negative breast cancers, particularly against the more aggressive and hormonal therapy-resistant ER-negative types. ' 2007 Wiley-Liss, Inc.