Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus.

Abstract

The role of tyrosine phosphorylation in the regulation of tyrosine protein kinase activity was investigated using site‐directed mutagenesis to alter the structure and environment of the three tyrosine residues present in the C terminus of avian pp60c‐src. Mutations that change Tyr 527 to Phe or Ser activate in vivo tyrosine protein kinase activity and induce cellular transformation of chicken cells in culture. In contrast, alterations of tyrosine residues present at positions 511 or 519 in c‐src do not induce transformation or in vivo tyrosine protein kinase activity. Amber mutations, which alter the structure of the pp60c‐src C terminus by inducing premature termination of the c‐src protein at either residue 518 or 523 also induce morphological transformation and increase in vivo tyrosine phosphorylation, whereas removal of the last four residues of c‐src by chain termination at residue 530 does not alter the kinase activity or the biological activity of the resultant c‐src protein. We conclude from these studies that C‐terminal alterations which either remove or replace Tyr 527 serve to activate the c‐src protein resulting in cellular transformation and increased in vivo tyrosine protein kinase activity.

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

“…The sequences of these putative protein kinases suggest a distinct form of activation, however, as they lack the phosphorylation site Thr Glu Tyr necessary for the activation of p42/44 forms of MAP/MBP kinases (Gonzalez et al, 1992 (Bryant and Parsons, 1982) and incubated at 41°C. Cells were routinely subcultured every 2 or 3 d. Transfection of cells with pRL-src, which is an infectious nonpermuted molecular clone of PrA RSV, results in the production of virus and a spread within the cell culture (Reynolds et al, 1987). Typically, 1 Mg of pRL DNA was used to transfect one 60-mm dish of CEF 24 h after trypsinization and plating.…”

“…High titer virus stocks were obtained from cells 10 d after transfection and stored at -70°C. Changes in cell morphology were routinely observed 6-8 d after transfection of pRL-v-src, pRL-c-527F (Reynolds et al, 1987), pRLtsl 19 (Bryant and Parsons, 1982), or pRL-dll 18C (Wang and Parsons, unpublished data). Replication of c-src and c-src mutant 2A-527F, which lack transforming activity, was assessed by resistance to superinfection with PrA RSV (Bryant and Parsons, 1982 40S subunits were prepared as described previously (Erikson et al, 1991).…”

The carboxy-terminal sequence of p56lck can regulate p60c-src.

“…Tyr-416 is the major site of phosphorylation in vitro (27,35). Enzymatically active forms of p60c-src are also phosphorylated at Tyr-416 in the cell (3,4,15,18,20,28,30). Tyr-527 is the major site phosphorylated in repressed forms of p60c-src in fibroblasts (7,19).…”

“…Transformation by the polyomavirus middle T antigen leads to dephosphorylation of Tyr-527 and stimulation of p60csrc kinase activity (2,4,11). Alteration of c-src codon 527 to specify Phe, a residue which is not phosphorylated, activates the transforming potential of c-src (3,18,28,30). Dephosphorylation of Tyr-527 in vitro results in a 10-to 20-fold stimulation of the specific activity of p60c-src in an in vitro kinase reaction (8).…”

“…The presence of the Ick sequence was confirmed by dideoxynucleotide sequencing (31). To provide an activated version of c-src as a positive control for transformation, the Tyr-527 codon of pMX was changed to code for Phe by using oligonucleotide-directed mutagenesis (3,18,28,30). The modified c-src genes were excised from the plasmids by using BamHI and BglII and cloned into the BamHI site of pLJ, a murine retroviral expression vector that carries a neomycin phosphotransferase minigene kindly provided by R. Mulligan (28).…”