The c-src protein isolated from neuronal cells (pp6Oc-src) displays a higher level of protein kinase activity than does pp6O-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60`rC expressed in neurons which could contribute to the enhanced activity of this form of pp6Oc-src (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src and (ii) a novel site(s) of serine phosphorylation. We characterized pp6oc-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation.The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp6Ocsrc+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60csrc and that pp6oc-src -expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60csrc could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain.The c-src gene has served as a model system in investigations of the function and importance of a large family of proto-oncogene products, the protein tyrosine kinases. High levels of the c-src protein (pp60c-src) have been identified in neural tissues (5,14,17,31,47,49), platelets (21), peripheral blood lymphocytes (21), monocytes (1, 18), and chromaffin cells (41). The majority of the c-src gene product expressed in neural tissues, however, is structurally distinct from the protein produced in nonneural tissues (4, 5) and is encoded by a uniquely processed c-src mRNA (4, 32, 37). Neuralspecific c-src cDNA clones isolated from both chicken brain (32) and mouse brain (37) cDNA libraries contain an 18-base-pair insertion located at the splice junction between exons 3 and 4 of the c-src gene. The additional six amino acids encoded by this miniexon are identical in avian and mammalian species, and this insertion is responsible for the altered electrophoretic mobility of the c-src protein in neuronal cells (designated pp6Oc-src ) (32, 37).The hexapeptide insert, located at amino acid 114 of avian pp60c-src (amino acid 117 of mammalian pp6OCs'C), lies outside the catalytic domain of pp6Ocsr', within a ...

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60csrc coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60cs1C. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60csrc termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon la, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites.