TOJ3, a target of the v-Jun transcription factor, encodes a protein with transforming activity related to human microspherule protein 1 (MCRS1).

Using the established quail cell line Q/d3 conditionally transformed by the v-jun oncogene, cDNA clones (TOJ2, TOJ3, TOJ5, TOJ6) were isolated by representational difference analysis (RDA) that correspond to genes which were induced immediately upon conditional activation of v-jun. One of these genes, TOJ3, is immediately and specifically activated after doxycycline-mediated v-jun induction, with kinetics similar to the induction of well characterized direct AP-1 target genes. TOJ3 is neither activated upon conditional activation of v-myc, nor in cells or cell lines non-conditionally transformed by oncogenes other than v-jun. Sequence analysis revealed that the TOJ3-specific cDNA encodes a 530-amino acid protein with significant sequence similarities to the murine or human microspherule protein 1 (MCRS1, MSP58), a nucleolar protein that directly interacts with the ICP22 regulatory protein from herpes simplex virus 1 or with p120, a proliferation-related protein expressed at high levels in most human malignant tumor cells. Similar to its mammalian counterparts, the TOJ3 protein contains a bipartite nuclear localization motif and a forkhead associated domain (FHA). Using polyclonal antibodies directed against a recombinant amino-terminal TOJ3 protein segment, the activation of TOJ3 in jun-transformed fibroblasts was also demonstrated at the protein level by specific detection of a polypeptide with an apparent M(r) of 65 000. Retroviral expression of the TOJ3 gene in quail or chicken embryo fibroblasts induces anchorage-independent growth, indicating that the immediate activation of TOJ3 in fibroblasts transformed by the v-jun oncogene contributes to cell transformation.

Suppression in transformed avian fibroblasts of a gene (CO6) encoding a membrane protein related to mammalian potassium channel regulatory subunits.

Gene expression patterns in normal and v-myc-transformed quail embryo fibroblasts were compared by mRNA differential display. Displaying approximately 2500 mRNA species by reverse transcription/PCR, reamplification of 73 differential cDNA fragments and rescreening by Northern analysis led to the isolation of a clone, termed CO6, that hybridized to an mRNA species present only in the normal but not in the transformed fibroblasts. Further analyses revealed that the 0.95-kb CO6 mRNA was present in all normal quail and chicken embryo fibroblasts tested, but that it was undetectable in a variety of established quail cell lines transformed by the v-myc, v-myc/v-mil, v-jun/junD or v-src oncogenes or by a chemical carcinogen. Furthermore, CO6 mRNA was not detectable in fibroblasts newly transformed by retroviral constructs carrying v-myc or v-jun alleles or by the avian sarcoma virus ASV17. In fibroblasts transformed by a temperature-sensitive v-src mutant, expression of CO6 was strongly induced at the non-permissive temperature and reduced at the permissive temperature. Nucleotide sequence analysis of quail CO6 cDNA indicated that the corresponding gene encodes a 200-amino acid protein with 46 to 48% amino acid sequence identity to the regulatory beta subunits (K(VCa)beta) of the bovine, human and canine high conductance Ca2+-activated K+ channels. No sequence homology to other ion channel subunits or to any other proteins in the databases was found. Like the K(VCa)beta subunits, the CO6 protein contains two putative transmembrane segments. Based on the relationship to mammalian K(VCa)beta both in primary structure and domain topology, the CO6 protein may represent the regulatory subunit of a yet unidentified avian Ca2+-activated potassium channel or a related membrane protein possibly involved in the regulation of cell proliferation.

The cysteine-rich protein family of highly related LIM domain proteins.

Here we describe a family of closely related LIM domain proteins in avian cells. The LIM motif defines a zinc-binding domain that is found in a variety of transcriptional regulators, proto-oncogene products, and proteins associated with sites of cell-substratum contact. One type of LIM-domain protein, called the cysteine-rich protein (CRP), is characterized by the presence of two LIM domains linked to short glycine-rich repeats and a potential nuclear localization signal. We have identified and characterized two evolutionarily conserved members of the CRP family, CRP1 and CRP2, in chicken and quail. Expression of the genes encoding both CRP1 and CRP2 is differentially regulated in normal versus transformed cells, raising the possibility that members of the CRP family may function in control of cell growth and differentiation.

Suppression in transformed avian fibroblasts of a gene (crp) encoding a cysteine-rich protein containing LIM domains.

Using cDNA subtraction and differential hybridization techniques, a cDNA library derived from normal quail embryo fibroblasts was screened for clones corresponding to genes whose expression was suppressed in v-myc-transformed, as compared with normal, quail embryo fibroblasts. One of the isolated cDNA clones corresponded to a 0.9-kb mRNA that was present in normal quail and chicken embryo fibroblasts, but was virtually absent from all transformed avian cells tested: quail embryo fibroblasts transformed by the v-myc, v-myc/v-mil or v-src oncogenes, cells derived from a methylcholanthrene-induced quail fibrosarcoma or v-myc-transformed chicken macrophages. Nucleotide sequence analysis of the original and supplementary cDNA clones indicated that the corresponding gene encodes a 194 amino acid cysteine-rich protein (M(r) 20,911). A database search revealed that the gene is the avian homolog of a human primary response gene (crp) of unknown function. Both the quail and human CRP proteins contain two copies of a cysteine-rich amino acid sequence motif (LIM) with putative zinc-binding activity that was previously identified in several proteins with presumed regulatory functions essential for cell growth or differentiation.