No abstract

Tyrosine protein kinases are important both in the normal regulation of cellular proliferation and in the oncogenic transformation of cells by several tumour viruses. The LSTRA Moloney murine leukaemia virus (M-MuLV)-induced thymoma cell line contains approximately 20-fold more phosphotyrosine in protein than do typical haematopoietic cell lines; this seems to result from the expression of an abnormally high level of a cellular tyrosine protein kinase termed p56tck (refs 3, 4). This kinase is normally expressed at low levels in most, but not all, murine T cells. The elevated levels of p56tck could contribute to the malignant properties of LSTRA cells. Therefore, we have isolated cloned complementary DNAs encoding the whole of p56tck. Sequence analysis shows it to be a novel cellular tyrosine protein kinase which is distinct from all others described to date. p56tck is encoded in LSTRA cells by a hybrid messenger RNA; approximately 200 nucleotides at the 5' end of the mRNA are identical to the 5' end of the genome of M-MuLV. The three- to ninefold transcriptional activation of the gene therefore results from retroviral promoter insertion.

A common DNA binding and dimerization domain containing an apparent "helix-loop-helix" (HLH) structure was recognized recently in a number of regulatory proteins, including the E47 and E12 proteins that bind to the KE2 motif in immunoglobulin X gene enhancer. The effect of site-directed mutagenesis on E47 protein multimerization and DNA binding was examined. Mutations in either putative helix domain disrupted protein dimerization and DNA binding. No DNA binding was observed when mutations were introduced in the basic region, but these mutants were able to dimerize. These basic region mutants were not able to bind to DNA as heterodimers with the wild-type E47 proteins, demonstrating that two functional basic regions are required for binding to DNA. Therefore the basic region mutants are "transdominant."The protein products of the E2A gene, E47 and E12, form homo-and heterodimers that recognize the KE2 site of the immunoglobulin K gene enhancer (1, 2). The KE2 site plays a critical role in the function of the enhancer (3,4). A DNA site similar to the KE2 sequence is also found in the insulin gene enhancer (5) and in the muscle creatine kinase gene enhancer (6), and both are important for the tissue-specific expression of their respective genes. The protein products of the three genes that induce myogenesis, MyoD (7,8), myogenin (9), and myf-5 (10) all share with E47 a highly conserved 60-amino acid stretch with predicted helix-loop-helix (HLH) structure (2) and act presumably by way of interaction with the KE2-like sequences in the muscle-specific genes. Other members ofthe "HLH family" have been defined based on amino acid sequence homology to the region of the E47 polypeptide required for specific recognition of DNA and include the myc family proteins; proteins important for Drosophila development, including the achaete-scute complex (11, 12), daughterless (13), twist (14), and Enhancer of split (15); and the lyl-l gene implicated in T-cell acute lymphoblastic leukemia (16). These proteins all contain a basic amino acid region followed by two short putative amphipathic helices separated by an intervening region that might form a loop, the HLH motif. In addition to homodimers, the HLH proteins can also form heterodimers that bind specifically to a common DNA sequence (2). Deletion mutagenesis analysis of the E47/E12 proteins showed that the basic region and HLH domain, which are located at the carboxyl terminus, are sufficient for the DNA binding activity in vitro (1, 2). To define further the regions required for specific contact with DNA and for dimer formation, as well as to determine whether the two can be functionally separated, site-directed mutagenesis of the E47 gene was performed. Experimental evidence is presented here that the amphipathic helices mediate complex formation between E47 proteins that results in the appropriate juxtaposition of the DNA binding region of the protein. Furthermore, dimerization is required for DNA binding, which also depends on a specific sequence of basic amino acids...