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Chromosome translocations involving band llq23 are associated with human acute leukemias. These translocations fuse the ALL-1 gene, homolog of Drosophila trithorax and located at chromosome band 11q23, to genes from a variety of chromosomes. We cloned and sequenced cDNAs derived from transcripts of the AF-4 and AF-9 genes involved in the most common chromosome abnormalities, t(4:11)(q21:q23) and t(9:11)(p22:q23), respectively. Sequence analysis indicates high homology between the AF-9 gene protein product and the protein encoded by the ENL gene fused to ALL-1 in (11:19) chromosome translocations. AF-4, AF-9, and ENL proteins contain nuclear targeting sequences as well as serine-rich and proline-rich regions. Stretches abundant in basic amino acids are also present in the three proteins. These results suggest that the different proteins fused to ALL-1 polypeptide(s) provide similar functional domains.

Ras (Ha-Ras). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein.The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP␥S⅐GST-HaRas. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP␥S⅐GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.Ras (Ha-Ras, Ki-Ras, N-Ras) is a signal-transducing guanine nucleotide-binding protein for tyrosine kinase-type receptors such as epidermal growth factor receptors and the Src family, leading to a mitogenic response and differentiation (for reviews, see Refs. 1 and 2). Ras has GDP-bound inactive and GTP-bound active forms, the latter of which makes physical contact with targets. Intensive investigations revealed that the Raf kinase family, consisting of c-Raf-1 (for reviews, see Refs. 3 and 4), A-Raf (5), and B-Raf (6 -9), is one of the direct targets for Ras. The activated Raf phosphorylates MAP 1 kinase kinase and activates it. Consequently the activated MAP kinase kinase activates MAP kinase, leading to the expression of certain genes such as c-fos (for reviews, see Refs. 10 and 11). Several molecules interacting with activated Ras in addition to Raf have been identified in mammals. These include phosphatidylinositol-3-OH kinase (12), Ral GDS (13,14), and Rin1 (15). On the basis of these observations, a variety of Ras targets may account for the pleiotropic functions of Ras. To understand the molecular mechanism of pleiotropic functions of Ras, it is essential to identify novel targets for Ras.In the present study, we discovered and partially purified another putative target for Ras with a molecular mass of about 180 kDa (p180) by use of GST-Ha-Ras affinity column chromatography and identified it as AF-6 (16), whose structure resembles that of Drosophila Canoe, which is involved in the Notch signaling pathway (17). S]methionine were purchased from DuPont NEN. A rabbit polyclonal antibody against a 16-mer peptide corresponding to 561-576 aa of human AF-6 (RVEQQPDYRRQESRTQ) was generated and purified. EXPERIMENTAL PROCEDURES Materials and Chemicals-AllPlasmid Construction-Plasmids, pGEX-Ha-Ras, pGEX-R-Ras, pGEX-RalA, and pGEX-RhoA were constructed as described previously (18). To obtain the in vitro translated N-terminal domain of AF-6 and Canoe, pRSET-AF-6 (36 -848 aa), pRSET-AF-6 (36 -206 aa), and pR-SET-Canoe (1-217 aa) were constructed as follows. The 2.4-kilobase cDNA fragment encoding AF-6 (36 -848 aa) was amplified by polymerase chain reaction from human brain Quick clone cDNA (Clontech Laboratories Inc., Palo Alto, CA). For the shorter N-terminal domain of ...

The ALL) gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to AF4 and AF9. The fused genes encode chimeric proteins. Because the Drosophila homologue ofALL), trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL) transcriptional activity may underlie its action in malignant transformation. To Chromosome 1 1q23 abnormalities are associated with 5-10% of human acute leukemias, in particular in children under the age of 1 year, and in secondary leukemias (1). These leukemias have a lymphoid, myeloid, or lymphoid-myeloid phenotype and uniformly carry a poor prognosis. More than 20 different 11q23 translocations have been identified by now (2, 3). In nearly all of these aberrations, the ALLI gene (also called HRX, MLL, or HTRX) is involved (4-7). ALLI is thought to be the human homologue of the Drosophila trithorax gene, which acts as a positive regulator of the homeotic gene complexes Antennapedia and Bithorax.11q23 chromosome translocations cleave ALL1 within the breakpoint cluster region and fuse it to a partner gene, resulting in production of fused RNAs and presumably of chimeric proteins (4, 5). The chimeric proteins containing the N terminus of ALL1 are thought to be the critical products of the translocations (for review, see ref. 8). Presently, 10 partner genes have been cloned (8-13).The mechanism(s) by which the chimeric ALL1 proteins act to trigger neoplasm is not known. Two models were proposed: the first (5) emphasized gain of function and suggested that the altered protein (specifically ALL1 /ENL) is a hybrid transcription factor in which the DNA binding domain of ALL1The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.localizes the hypothetical transactivation domain of ENL to regulatory sites of target genes. The second (14) suggested that the cleavage of ALL1 protein results in loss of its function and that the partner polypeptide augments this loss. Resolution between the two models would necessitate developing biological and biochemical assays to measure transcriptional regulatory activity of the normal ALL1 protein, of the normal partner proteins, and of the chimeric species. As a first step in this direction, we looked for domains with transcriptional activation or repression potential within ALL1, within the AF4 and AF9 partner proteins (presumed to be nuclear proteins), and within the AF17 partner polypeptide, which contains a dimerization motif. A similar approach to investigate the ALL1 protein was recently reported (15). MATERIALS AND METHODSPlasmids. Effector plasmid vectors were either RSV-GAL4 (16) or pGALM (17). The former contains a GAL4 DNA binding domain (amino acids 1-147) flanked by the Rous sarcoma virus (RSV) promoter and ...

Chromosome region 11q23 is involved in reciprocal chromosome translocations associated with human acute leukemias. These aberrations fuse theALL-i gene located at llq23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. Here we report the cloning and characterization of the ALL-I partner at 17q21, the AF17 gene. The AF17 gene encodes a protein of 1093 amino acids, containing a leucinezipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus. The latter can be arranged in three zinc fingers and shows homology to a domain within the protein Brl4O (peregrin). AF17 contains stretches of amino acids previously associated with domains involved in transcriptional repression or activation. Based on features of AF17 and of the proteins encoded by the other partner genes analyzed and in conjunction with other recent studies, we propose a model in which ALL-1 rearrangements result in loss of function of the gene. In this model, the partner polypeptide plays an accessory role either by repressing activity of the truncated ALL-1 protein or by blocking the function of the normal protein presumably present in the leukemic cells.