Differential expression of p62c-yes in normal, hyperplastic and neoplastic human epidermis

The protein product of the c-yes proto-oncogene, p62c-yes, is highly expressed in a variety of mammalian cell types, including neurons, spermatozoa, platelets, and epithelial cells. In order to understand the function of p62c-yes in epithelial cells, the expression and localization of p62c-yes was studied in cultured human epidermal keratinocytes and in normal, hyperplastic, and neoplastic human epidermis. Human keratinocytes in culture produce a single 4kb c-yes mRNA and a 62kd protein product, p62c-yes, which is active as a protein tyrosine kinase. Using affinity-purified antibodies generated to the amino-terminus of the human c-yes protein, the expression of p62c-yes was localized to keratinocytes in the basal epidermal layer of normal neonatal and adult epidermis. There was a marked reduction in expression of p62c-yes by suprabasal keratinocytes undergoing progressive differentiation. By immunofluorescence microscopy, p62c-yes was localized to the plasma membrane and to a perinuclear cytoplasmic area in cultured keratinocytes. The apparent association of p62c-yes with plasma membranes was particularly evident in suprabasal keratinocytes from hyperplastic epidermis. Neoplastic keratinocytes in basal cell carcinomas showed a marked reduction in p62c-yes expression compared to normal basal keratinocytes in epidermis or to proliferating cultured keratinocytes. Thus the expression of p62c-yes by one epithelial cell type, the keratinocyte, is altered by cellular differentiation and neoplastic transformation. Keratinocytes provide a normal epithelial cell model in which the biochemical function of p62c-yes can be studied.     

c-yes protein kinase is associated with a 38 kD protein in cerebellum

p62c-yes, the protein product of the yes proto-oncogene, was found in association with a cellular protein of 38 kD in chicken cerebella. The complex was detected by immunoprecipitation of cerebellar membranes with affinity purified anti-yes IgG followed by in vitro phosphorylation of the immunocomplex. Both proteins were found to be phosphorylated exclusively on tyrosine. The sedimentation profile of the yes kinase indicated that a fraction of p62c-yes was complexed with the 38 kD protein and comigrated in the gradient with a molecular mass of approximately 150 kD. We have previously described the association of p60c-src with a 38 kD protein, referred to as p38 [Grandori, C. and Hanafusa, H., J. Cell Biol. (1988), 107: 2125-2135]. Comparison of the src-associated p38 with the yes-associated 38 kD protein indicates that they are indistinguishable by one-dimensional peptide mapping. Association of p38 with more than one member of the src-family of tyrosine kinases makes this protein an attractive probe to study the structural and functional aspects of these enzymes.     

The transforming activity of the chicken c-myc gene can be potentiated by mutations

It was previously demonstrated that four different avian v-myc oncogenes harbor several point mutations. At least one of these leads to an amino acid substitution located in the proximity of position 61 in the second exon, whereas additional substitutions are found in exon 3. We have investigated whether these mutations affect the transforming activity of myc. By constructing avian retroviral genomes expressing hybrid gag-myc oncogenes, in which all or parts of the v-myc domains were replaced by corresponding parts of c-myc, we show here that a substitution of threonine 61 of c-myc for a methionine (as in v-mycmc29) significantly enhances the fibroblast transforming capacity of the recombinant oncogene. However, such a hybrid v/c-myc gene is still several fold less active than the v-mycmc29 oncogene. We have also expressed c-myc from subgenomic retroviral mRNAs: in these constructions the AUG of gag in the RNA leader sequence is in the same reading frame as that of c-myc, apparently leading to the production of a myc protein with 11 N-terminal amino acids encoded by gag and non-coding c-myc sequences. These myc proteins also transform chicken embryo fibroblasts, albeit with a lower efficiency than v-myc, again suggesting that mutations can increase the transforming capacity of myc.     

Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine

Antibodies to phosphotyrosine were used to identify proteins phosphorylated on tyrosine in chicken embryo fibroblasts transformed by the oncogenes, v-src, v-yes, v-fps, v-ros, or v-erb-B. These antibodies allowed detection of a minimum of 50, 44, and 47 bands in immunoblots of cellular lysates from fibroblasts transformed by v-src, v-yes, or v-fps respectively. Eight of these bands were also detected in fibroblasts transformed by v-ros and v-erb-B, suggesting that the cellular transformation induced by v-ros, v-erb-B, v-src, v-yes, and v-fps may be achieved by the phosphorylation of some of the same proteins. The viruses SD10 and SD11 are point mutants of the Prague-C strain of Rous sarcoma virus that encode non-myristylated p60v-src. They do not induce the transformation of chicken fibroblasts. Approximately thirty bands were detected by antibodies to phosphotyrosine in fibroblasts transformed by wild-type Prague-RSV-C, only four of which were not substrates nonmyristylated mutant p60v-src. This suggests that the phosphorylation of a large subset of the substrates of p60v-src is not sufficient for transformation.     

Localization of p62c-yes protein in mammalian neural tissues

Expression of the c-yes proto-oncogene in mammalian brain, retina and adrenal gland was studied by immunohistochemistry and immune assays with affinity-purified anti-yes IgG. Immunohistochemical staining with anti-yes IgG showed that in the central nervous system p62c-yes is highly expressed in mitral cells of the olfactory bulb, Purkinje cells of the cerebellum, hippocampal neurons and granule neurons of the dentate gyrus and ependymal cells lining central nervous system ventricles. Less intense labeling by anti-yes IgG was observed in most neuronal cell bodies in the brain. In addition, we observed intense c-yes immunoreactivity in the ganglion cells of the retina, the inner segment layer of rods and cones and medullary cells of the adrenal gland. Mapping p62c-yes expression to specific areas of mammalian neural tissues points to attractive experimental systems which could be used to investigate the function of the proto-oncogene product in neural processes.     

Myc protein structure: localization of DNA-binding and protein dimerization domains

Wildtype and mutant v-Myc proteins were overexpressed in Escherichia coli using the T7 RNA polymerase system, and the in vitro DNA-binding activities of partially or highly purified proteins were analysed by native DNA-cellulose chromatography. For the construction of the expression plasmids, cloned proviral DNA from wildtype MC29 or from its spontaneous deletion mutant Q10C was used, the latter lacking internal v-myc sequences. Both the wildtype (p59) and the mutant (p42) recombinant protein contain at their amino termini 12 amino acids encoded by the vector, followed by 11 gag amino acids and 9 amino acids encoded by v-myc sequences derived from noncoding c-myc sequences. In addition, p59 contains 416 amino acids encoded by v-myc sequences derived from the complete chicken c-myc coding region, whereas p42 lacks 120 amino acids from the central region of the Myc protein including the highly acidic domain. Two additional proteins were engineered which contain the first 309 (p53) or the last 107 (p16) amino acids, respectively, of the Myc protein sequence in addition to vector-encoded amino acids. The p16 protein represents the carboxyl terminus of the Myc protein sequence containing both a muscle determination gene (MyoD1) homology region, including a basic motif and an amphipathic helix-loop-helix motif, and a leucine heptad repeat. All proteins, except p53 which lacks the carboxyl-terminal Myc protein sequences, bound to native DNA-cellulose and were eluted with 200-500 mM NaCl. Based on the DNA-binding activities of recombinant or spontaneous mutant v-Myc proteins extracted from bacterial or from transformed avian cells, we conclude that the DNA-binding domain of avian Myc proteins is confined within the last 86 carboxyl-terminal amino acids. The same region is also shown to be necessary and sufficient for Myc protein dimerization. This 86-amino acid region essentially encompasses a putative basic DNA contact surface and a tandem array of two presumed protein dimerization motifs, helix-loop-helix and leucine repeat.     

Regional mapping of the human proto-oncogene c-yes-1 to chromosome 18 at band q21.3

Human proto-oncogene c-yes-1 homologous to the viral oncogene (v-yes) of an avian Y73 sarcoma virus was mapped to region q21.3 of chromosome 18 by in situ hybridization. This finding confirmed and more rigidly substantiated the previous assignment which was based on analyses of human X mouse somatic cell hybrids and Southern blots.     

The avian cellular homolog of the oncogene jun

We have isolated chicken genomic and cDNA clones representing the jun oncogene of avian sarcoma virus 17 (ASV17). The genomic clone lacks intron sequences within its protein coding domain, contains a CAAT box, seven SP-1 consensus sequences and TATA box-like elements upstream and two poly(A) addition signals downstream of the coding domain. The cellular jun protein is 310 amino acids in length. Cellular and viral jun proteins differ by three nonconservative amino acid substitutions of which two are located in the DNA-binding domain, by a 27-amino-acid deletion in the amino terminal third of the viral jun protein, by eleven cell-coded amino acids that link the cellular jun coding domain to the viral gag domain and by the partial gag sequences constituting the amino terminal of the viral gag-jun fusion protein. The availability of a cellular jun cDNA now allows the construction of reciprocal recombinants between the viral and the cellular gene which will define the structural features required for the oncogenicity of v-jun.     

Phosphatidylinositol-3 kinase is activated in v-src, v-yes, and v-fps transformed chicken embryo fibroblasts.

PI-3 kinase activity has been shown to associate with p60v-src. We found that immunoprecipitates of p60v-src exhibit an activity that catalyzes the formation of PI-3-P, PI-3,4-P2 and PIP3 from PI, PI-4-P, and PI-4,5-P2, respectively. Transformation of chicken embryo fibroblasts (CEF) by p60v-src of Rous sarcoma virus (RSV) caused elevation of PI-3-P, PI-3,4-P2, and PIP3, suggesting that the PI-3 kinase may be activated in these cells. Similar elevations were seen in cells transformed with the v-yes or v-fps oncogenes, but not with v-ros or v-ras. We have established also a system that allows the binding of PI-3 kinase to purified p60v-src in vitro, reproducing the binding seen in vivo. This assay indicated that more PI-3 kinase activity binds to purified p60v-src in cell lysates from CEF transformed with v-yes or v-fps, suggesting that some modification or over-expression of PI-3 kinase takes place in these cells.     

Isolation and sequencing of cDNA clones homologous to the v-fgr oncogene from a human B lymphocyte cell line, IM-9

Two c-fgr cDNA clones were isolated from a cDNA library derived from a human B lymphocyte cell line, IM-9. Sequence analysis of the clones showed that they contained inserts corresponding to nearly full-length human c-fgr mRNA, which could encode a polypeptide of 529 amino acids with a calculated molecular weight of 59,478. Although the amino acid sequence between Gly-78 and the carboxy-terminus of the c-fgr is highly homologous to the corresponding sequence of the c-yes protein, the homology between the two proteins is low in the amino-terminal proximal region. Northern blot hybridization analysis using the c-fgr specific sequence showed that the c-fgr mRNA was expressed at higher level in the liver than in the brain, lung, or kidney of a human fetus.     

Amplification of c-yes-1 proto-oncogene in a primary human gastric cancer

Abnormalities of cellular oncogenes in 22 cases of human primary gastric cancer were screened by Southern blot analysis using 16 onc probes. Human cellular sequences related to the v-yes oncogene of Y73 avian sarcoma virus (human c-yes-1 gene) were found to be amplified in a primary gastric cancer. The degree of amplification was about 4- to 5-fold. Another v-yes-related cellular gene (human c-yes-2, a pseudogene) was not amplified in this tumor. The normal stomach tissue adjacent to the tumor tissue in the same patient showed no amplification of c-yes-1 gene, suggesting that the amplified c-yes-1 is involved in the onset or the progress of this carcinoma.     

Binding to nucleophosmin determines the localization of human and chicken ARF but not its impact on p53

The ARF tumour suppressor gene encodes a small highly basic protein whose known functions are largely determined by the amino acids encoded within the first exon. In mammals, the protein incorporates additional residues specified by an alternative reading frame in the second exon of INK4a, but this arrangement does not apply to the chicken homologue. In exploring the intracellular localization of chicken p7(ARF), we found that while the FLAG- and HA-tagged versions localize in the nucleolus, in line with mammalian ARF, the GFP-tagged version is excluded from the nucleolus. Here we show that irrespective of the source or composition of the ARF fusion proteins, versions that accumulate in the nucleolus share the ability to bind to nucleophosmin (NPM). Depletion of NPM with siRNA results in the re-location and destabilization of nucleolar forms of ARF but has little effect on the location or stability of a nucleoplasmic form of ARF. Importantly, knockdown of endogenous NPM does not impair the ability of ARF to bind to MDM2 and stabilize p53. These findings support the view that nucleolar localization determines the stability of ARF but not its primary function.