Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Abstract

Because functionally significant substrates for the tyrosyl protein kinase activity of pp6)v-slC are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp6OV-slc altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-y, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp6OV-sC and the EGF receptor, phospholipase C-,y was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60's'-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.The v-src oncogene product, pp60v-sr, is a 60-kDa tyrosyl protein kinase, and this enzymatic activity is essential to its ability to cause malignant transformation (52). Numerous cellular proteins become phosphorylated on tyrosine in src-transformed cells (9,34,35,45), but the mechanism(s) by which these src-induced phosphorylations alter cellular behavior is unknown. Studies utilizing mutant forms of pp60v-src which retain kinase activity but are partially defective in their ability to cause phenotypic transformation suggest that only a limited subset of these tyrosine-phosphorylated proteins play an essential role in cellular transformation by src (10,33,35,39,57). It is widely suspected that the physiologically important substrates for pp60v-src will prove to be proteins which play a role in normal growth regulation, although it has been difficult to identify pp60v-src_ induced changes in phosphorylation and function of such regulatory proteins. It also is generally believed that at least some critical substrates for pp60v-sr" are associated with cell membranes, since cytosolic variants of pp60v-src are unable to transform cells (33,39,52,57).The best-characterized membrane-associated growth regulatory proteins are the transmembrane receptors for growth factors, and we have hypothesized (49) that pp60v-src might alter cellular regulation by phosphorylating and activating growth factor receptors. Indeed, we recently found that a 95-kDa cellular glyco...

Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

“…For example, EGF/EGFR induces the activation of c-Src in several types of cells (Osherov & Levitzki 1994;Sato et al 1995a;Weernink & Rijksen 1995). On the other hand, over-expression of c-Src results in an augmentation of EGF-responsiveness of cells (Luttrell et al 1988) and EGFR/kinase activity (Wasilenko et al 1991). In addition, it has been shown that c-Src activity is required for the up-regulation of EGFR in lysophosphatidic acid-treated cells (Daub et al 1997).…”

The interplay between Src family kinases and receptor tyrosine kinases

“…In addition to these autophosphorylation sites, however, SFK activation can lead to additional tyrosine phosphorylation of EGF-R and to activation of EGFdependent signaling. For example, coexpression of EGF-R and of a temperature-sensitive mutant of v-Src in Rat-1 fibroblasts induces rapid tyrosine phosphorylation of two non-autophosphorylation sites in EGF-R (Wasilenko et al, 1991). Further studies using purified wild-type or kinase-dead EGF-R and c-Src identified three potential sites responsive to c-Src phosphorylation in vitro: Tyr 1148 , Tyr 1173 , and possibly Tyr 703 (Wright et al, 1996).…”

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

“…There is no indication that calpactin II would become tyrosine phosphorylated in v-src transformed cells (T. Hunter, personal communication), and thus it is not likely a component of the transformation-specific tyrosinephosphorylated 36 kD band. However, it should be kept in mind that calpactin II is an identified substrate for the EGF receptor tyrosine kinase (55) that becomes activated upon v-src transformation (62). The physiological function of calpactins is still unsettled, but they are supposed to be important for cytoskeletal organization.…”