Generation and characterization of transforming variants of the lck tyrosine protein kinase.
p56lck, a tyrosine protein kinase of the src family, is overexpressed in two murine thymoma cell lines, LSTRA and Thy19, as a result of the integration of Moloney murine leukemia virus sequences upstream of the lck gene. The majority of the p56lck in these cell lines is translated from a hybrid mRNA comprised of the 5' untranslated region of the murine leukemia virus env mRNA and lck coding sequences. The retroviral promoter giving rise to this transcript has been molecularly cloned. To examine whether overexpression of unmutated p56lck might induce cellular transformation, we constructed a plasmid in which the murine leukemia virus promoter from LSTRA cells directed the expression of p56lck. This construct gave rise to foci when transfected into rat 208F fibroblasts. Cells from many of the foci also grew in soft agar. Tryptic peptide mapping showed that the p56lck in the transformed cells was phosphorylated at Tyr-394, the autophosphorylation site, but not detectably at Tyr-505, an inhibitory site. Because an antiserum made to the carboxy terminus of p56lck could not immunoprecipitate p56lck from these transformed cells, the possibility arose that the proteins expressed in the transformed fibroblasts contained mutations that altered the carboxy terminus of the protein. cDNAs derived from the 3' ends of the lck mRNAs in two of the foci were cloned, and both were found to be derived from an lck gene that was truncated upstream of the codon for Tyr-505 and fused to random sequences derived from other parts of the construct used for transfection. lck therefore resembles several other src family members in that it can be rendered oncogenic by replacement of the region encoding the inhibitory, carboxy-terminal phosphorylation site by random amino acid sequences.
Adler
H T
HT
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92186.
Sefton
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D015870:Gene Expression; D014644:Genetic Variation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009857:Oncogenes; D010449:Peptide Mapping; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013945:Thymoma; D013953:Thymus Neoplasms; D014162:Transfection; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)