Cloning and expression of a chicken c-rel cDNA: unlike p59v-rel, p68c-rel is a cytoplasmic protein in chicken embryo fibroblasts.
We isolated and sequenced a 3727 bp clone of the c-rel proto-oncogene from a chicken embryo fibroblast (CEF) cDNA library. Sequence comparison to the retroviral oncogene v-rel showed conclusively that the v-rel protein is truncated at both the amino- and carboxy-termini as compared to the c-rel protein. In vitro transcription and translation of this clone yielded a 68,000 dalton polypeptide that co-migrated on SDS polyacrylamide gels with p68c-rel from avian spleen cells. We inserted this c-rel cDNA clone into an avian retroviral vector (pJD214c-rel), and over-expressed p68c-rel in CEF. Over-expression of p68c-rel did not induce morphological transformation of these cells. Unlike p59v-rel, which is a nuclear protein in CEF, indirect immunofluorescence showed that p68c-rel in JD214c-rel infected CEF is located exclusively in the cytoplasm of these cells, even though the sequence of p68c-rel showed that it contains a nuclear localizing sequence identical to the one previously identified in p59v-rel. Furthermore, the c-rel protein does contain a nuclear localizing sequence which can function in CEF since replacement of the v-rel nuclear localizing sequence with the homologous domain from c-rel resulted in a hybrid rel protein that was located in the nucleus in CEF. Mutant c-rel proteins, deleted of the carboxy-terminal sequences not present in p59v-rel, localized to the nucleus in CEF. Our results show that the carboxy-terminus of p68c-rel inhibits nuclear localization of the protein, and suggest that subcellular location may be a form of regulation of the activity of p68c-rel.
Capobianco
A J
AJ
Biology Department, Boston University, Massachusetts 02215.
Simmons
D L
DL
Gilmore
T D
TD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification; D020569:Proto-Oncogene Proteins c-rel; D011519:Proto-Oncogenes; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D020569:Proto-Oncogene Proteins c-rel; D011505:Protein-Tyrosine Kinases