ABSTRACTcDNAs representing the a subunit of polyomavirus enhancer binding protein 2 (PEBP2; also cafled PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion 13 subunit. The human AMLI gene related to t(8;21) acute myeloid leukemia also has a run homology region. Together with the 18 subunit, which increases the affinity of the a subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2a mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is,involved in regulation of T-ceil-specific gene expression.Potential use of polyomavirus (Py) as a probe of mouse development has been recognized since it was observed that the wild-type Py did not grow in embryonal carcinoma cells but that it grew when the cells were induced to differentiate (1)(2)(3)(4)(5). Through the analysis of the Py enhancer, which determines the differentiation-dependent and cell-typespecific expression of the viral genes, a number of transcription factors involved in developmental regulation have been identified (6-10).PEBP2/PEA2, which binds to both the A and B cores of the Py enhancer (11), is undetectable in embryonal carcinoma F9 cells and becomes detectable after the cells are induced to differentiate (6,7). It specifically recognizes a consensus sequence, R/TACCRCA (R, purine), which was originally identified in the Py enhancer (11) and is also compatible with the core motif of murine leukemia virus enhancers (12,13). In addition, many T-cell-specific genes, such as T-cell receptor (TCR) a, X3, yand 8 and CD3e genes, contain potential PEBP2 binding sites, suggesting the possibility that PEBP2 is involved in T-cell-specific gene expression (13 [708][709][710][711][712][713][714]. The mutations are the same as the M2 mutation (14). For expression in mammalian cells, the coding regions of al, a2, and 831 cDNAs were cloned into the Xho I site of pCDMPy, in which the Py origin and enhancer region were deleted from pCDM8 (Invitrogen), resulting in pCDMPy-al, pCDMPy-a2, and pCDMPy-31. The bacterial expression plasmids pETal and pETa2, carrying the whole coding regions of al and a2 cDNAs, were constructed in the same way as pETf82 (16). To produce deletion mutants of a2, the whole coding region of a2 cDNA was inserted into the BamHI/HindIII fragment of plasmid pQE9 (Qiagen) resulting in pQE9-a2 encoding a fusion protein tagged with an N-terminal histidine cluster. Various deletions were introduced into pQE9-a2 using appropriate restriction sites: N94C306 encodes a region of aa 94-306; N1C226 encodes aa 1-226; N1C158 encodes aa 1-158; N80C226 encodes aa 80-226. N1C226, N1C158, and N80C226 proteins have one, two, o...

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Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.

Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.

A murine transcription factor, PEBP2, is composed of two subunits, a and ,B. There are two genes in the mouse genome, PEBP2aA and PEBP2&aB, which encode the at subunit. Two types of the aLB cDNA clones, aLBi and aB2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8-and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described aB protein referred to as oiBl, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed aB2, which is identical to aBl except that it has an internal deletion of 64 amino acid residues. Both otBl and aB2 associate with PEBP2,I and form a heterodimer. The atB2/4 complex binds to the PEBP2 binding site two-to threefold more strongly than the aB1/,I complex does. aBl stimulates transcription through the PEBP2 site about 40-fold, while aB2 is only about 25 to 45% as active as aBl. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that aA, aB, and ,B genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AMLI on chromosome 21q22 and PEBP24/CBFD on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that the previously described chimeric gene products, AML1/MTG8(ETO) and AMLl-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AMLL.A murine transcription factor, PEBP2, also called PEA2, was identified as a factor which binds to the region of the polyomavirus enhancer originally termed the PEA2 site (35). PEBP2 functionally cooperates with other enhancer corebinding proteins, including AP1 and Ets family proteins, PEA3 (48), , and PEBP5 (2), and plays an important role in stimulation of transcription and viral DNA replication. PEBP2 is composed of a and 3 subunits (32, 34). The at subunit bind to DNA by recognizing the consensus sequence, (Pu/T) ACCPuCA (13,34). The companion 3 subunit binds to the cx protein and increases the affinity of the a protein to DNA without binding to DNA by itself (32). cDNAs coding for both subunits have been cloned and characterized in detail (32, 34).PEBP2 has recently been shown to be closely related to acute myeloid leukemia (AML). The t(8;21)(q22;q22) translocation is one of the most frequent chromosome abnormalities in AML and is classified into the FAB-M2 subtype of AML (39). It has been shown that the t(8;21) breakpoints in the chromosome 21 are clustered in the recently described AMLI gene (27), the product of which shares a high homology within the 128-amino-acid (aa) region with the product of the Drosophila segmentation gene, runt (14), and the at subunit of PEBP2 (8,9,34 genome, oaB, which shares a high sequence homology with the original ao gene, termed aA. The aB gene as a whole is highly homologous to human AMLl (3). The discovery of the homology of the AML1 protein to the otsubunit o...

A member of the polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is composed of PEBP2␣B1/AML1 (as the ␣ subunit) and a ␤ subunit. It plays an essential role in definitive hematopoiesis and is frequently involved in the chromosomal abnormalities associated with leukemia. In the present study, we report functionally separable modular structures in PEBP2␣B1 for DNA binding and for transcriptional activation. DNA binding through the Runt domain of PEBP2␣B1 was hindered by the adjacent carboxyterminal region, and this inhibition was relieved by interaction with the ␤ subunit. Utilizing a reporter assay system in which both the ␣ and ␤ subunits are required to achieve strong transactivation, we uncovered the presence of transcriptional activation and inhibitory domains in PEBP2␣B1 that were only apparent in the presence of the ␤ subunit. The inhibitory domain keeps the full transactivation potential of full-length PEBP2␣B1 below its maximum potential. Fusion of the transactivation domain of PEBP2␣B1 to the yeast GAL4 DNA-binding domain conferred transactivation potential, but further addition of the inhibitory domain diminished the activity. These results suggest that the activity of the ␣ subunit as a transcriptional activator is regulated intramolecularly as well as by the ␤ subunit. PEBP2␣B1 and the ␤ subunit were targeted to the nuclear matrix via signals distinct from the nuclear localization signal. Moreover, the transactivation domain by itself was capable of associating with the nuclear matrix, which implies the existence of a relationship between transactivation and nuclear matrix attachment.The polyomavirus enhancer binding protein 2 (PEBP2), also called core binding factor (CBF), is a transcription factor complex composed of ␣ and ␤ subunits (reviewed in references 21 and 51). The ␣ subunit binds to DNA and harbors the transactivating activity, while the ␤ subunit enhances the DNA binding activity of the ␣ subunit. In mammals, members of the ␣ subunit family are encoded by three genes, PEBP2␣A/ CBFA1/AML3, PEBP2␣B/CBFA2/AML1, and PEBP2␣C/ CBFA3/AML2, and all belong to the Runt domain gene family, which includes the Drosophila genes runt and lozenge. The ␤ subunit is encoded by a single gene, PEBP2␤/CBFB, whereas two genes, brother and big brother have been identified in Drosophila.Among the three mammalian ␣ subunit genes, PEBP2␣B/ CBFA2/AML1 (2, 36, 51) is disrupted in chromosomal translocations associated with several types of leukemia, including the M2 subtype of the French-American-British classification of leukemia, which is characterized by the 8-to-21 chromosome translocation [t(8;21)], and childhood acute lymphoblastic leukemia with the associated t(12;21) translocation. The t(8;21) and t(12;21) translocations produce the chimeric proteins, AML1/ETO(MTG8) and TEL-AML1, respectively (13,19,37). These proteins retain the entire Runt domain in their PEBP2␣B/AML1 portions, which is essential and sufficient for dimerization with the ␤ subunit and for DNA binding. In addition, it is...

Each of the two human genes encoding the ␣ and ␤ subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the ␣ and ␤ subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2␣ and -␤ cDNAs revealed that the full-size ␣A1 and ␣B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the ␤ subunit is localized to the cytoplasm. Deletion analysis demonstrated that there are two regions in ␣A1 responsible for nuclear accumulation of the protein: one mapped in the region between amino acids 221 and 513, and the other mapped in the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size ␣A1 and ␤ proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N-or C-terminally truncated ␣A1 proteins devoid of the region upstream or downstream of the Runt domain colocalized with the ␤ protein in the nucleus. In these cases, the ␤ protein appeared to be translocated into the nucleus passively by binding to ␣A1. The chimeric protein containing the ␤ protein at the N-terminal region generated as a result of the inversion of chromosome 16 colocalized with ␣A1 to the nucleus more readily than the normal ␤ protein. The implications of these results in relation to leukemogenesis are discussed.

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