Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Abstract

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, are transformed at 36 degrees C, but at 40 degrees C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v-src product of the virus' transforming src gene. At 40 degrees C, these tsLA23-NRK cells were arrested in G1/G0 by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet-derived growth factor (PDGF) at 40 degrees C, or (2) transformed cells by lowering the temperature to a pp60v-src-activating 36 degrees C without adding exogenous growth factor(s). The level of pp60v-src kinase activity rose dramatically in these serum-deprived cells within 30 minutes of lowering the temperature to the permissive 36 degrees C, and it fell just as rapidly when the cells were returned to the restrictive 40 degrees C. As little as a 2-hour exposure to 36 degrees C, with an attendant 2-hour burst of pp60v-src kinase activity, was enough to stimulate serum-deprived tsLA23-NRK cells to transit G1 and initiate DNA replication, but not to divide. Much more prolonged pp60v-src activity was needed for these serum-deprived cells to complete their cycle and divide. The prereplicative development of quiescent tsLA23-NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v-src-stimulated cells progressed through G1 and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v-src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v-src were produced with comparable efficiency, although it took longer for pp60v-src-stimulated cells to translate these transcripts and to initiate DNA replication, probably because of their unstimulated protein-synthetic machinery.

A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

“…These two cell lines were also able to proliferate in plasma-derived, as well as, wholeblood derived serum. Similar results have been reported for NRK cells expressing the src oncogene (Chen et al, 1977;Durkin and Whitfield, 1984).…”

“…Similarly, most transformed cells are anchorage-independent, have a greatly reduced requirement for exogenous growth factors, and lose contact inhibition. For example, these three properties are lost in parallel when NRK fibroblasts are transformed by K-ras (see above), v-mos (see above), or v-src (Chen et al, 1977;Durkin and Whitfield, 1984). The reduced requirement for exogenous growth factors likely results, at least in part, from the fact that many transformed cells secrete mitogenic growth factors that stimulate cell proliferation in an autocrine fashion (reviewed in Pardee, 1989).…”

Characterization of G1 transit induced by the mitogenic-oncogenic viral Ki-ras gene product.

“…The points are means ± SEM of four cultures. In many of these respects, the viral p21 ras protein is like the pp6Ov-src tyr protein kinase of avian sarcoma virus (14).…”

“…For example, the v-sis gene product is structurally similar to platelet-derived growth factor (12, 36), and the v-erbB product is homologous to part of the epidermal growth factor receptor (13). Moreover, the product of the v-src gene, pp60v-src, is a complete mitogen, the activity of which is independent of external growth factors in serum (14,15).Oncogenic members of the ras family of oncogenes and their ras protein products have been shown to activate the proliferative machinery when microinjected into quiescent cells (1,16,30). The ras proteins are membrane-associated, GTP-binding GTPases, the oncogenic forms of which differ from the nononcogenic forms usually by having amino acid substitutions at position 12 as well as position 59 or 61 and reduced GTPase activity (18,25,31).…”

“…For example, the v-sis gene product is structurally similar to platelet-derived growth factor (12, 36), and the v-erbB product is homologous to part of the epidermal growth factor receptor (13). Moreover, the product of the v-src gene, pp60v-src, is a complete mitogen, the activity of which is independent of external growth factors in serum (14,15).…”