Cortactin, a p80/85 protein ®rst identi®ed as a src kinase substrate, is thought to be involved in the signaling pathway of mitogenic receptors and adhesion molecules mediating cytoskeletal reorganization. The cortactin gene, EMS1, maps to chromosome 11q13, a region ampli®ed in head and neck squamous cell carcinomas (HNSCC) and breast cancer, which display lymph node metastasis and an unfavorable clinical outcome. To further address the role of cortactin in the malignant phenotype of cells, we stably overexpressed cortactin in NIH3T3 ®broblasts and evaluated the e ects of elevated cortactin on cellular proliferation, motility and invasiveness. Cortactin overexpressing cells did not display any striking morphological changes, nor any signi®cant di erences in cell proliferation or saturation density as compared to control NIH3T3 cells. Furthermore, the cortactin overexpressing cells were anchorage dependent for growth. Interestingly, cortactin overexpressing cells were more motile and invasive in modi®ed Boyden chamber assays. These results suggest that overexpression of cortactin may play a role in tumor progression by in¯uencing tumor cell migration and invasion.

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...

o 1996 Wiley-Liss, Inc.

The tyrosine kinase pp60v-src, encoded by the v-src oncogene, seems to regulate phosphatidylinositol metabolism. The effect of pp60v-src on control points in inositol phosphate production was examined by measuring the amounts of inositol polyphosphates in Rat-1 cells expressing wild-type or mutant forms of the protein. Expression of v-src-resulted in a five- to sevenfold elevation in the steady-state amount of an isomer of inositol tetrakisphosphate, whereas the concentrations of inositol trisphosphates or other inositol tetrakisphosphates were not affected. The activity of a key enzyme in the formation of inositol tetrakisphosphates, inositol (1,4,5)-trisphosphate 3-kinase, was increased six- to eightfold in cytosolic extracts prepared from the v-src-transformed cells, suggesting that this enzyme may be one target for the pp60v-src kinase and that it may participate in the synthesis of novel, higher order inositol phosphates.

No abstract

Malignant tumor cells exhibit a number of distinct properties involved not only with deregulated cell proliferation but also enhanced migration and invasion. The Jun oncogene has been well studied in regard to its role in cell proliferation. Many of the target genes deregulated by Jun encode matrix metalloproteases (MMPs) such as MMP1, MMP3 and MMP9. These targets implicate a prominent role for Jun in tumor cell invasion, in addition to its role in growth transformation. To investigate this possibility, we have examined the effect of over-expression of transforming and non-transforming versions of Jun on motility and invasion of chicken embryo fibroblasts (CEFs). We found that over-expression of either form of Jun results in elevated intrinsic cellular motility as well as increased motility in response to several different chemoattractants, including 3T3-conditioned media, basic fibroblast growth factor, hepatocyte growth factor and Matrigel. The capacity of these cells to invade through Matrigel is also elevated as a result of Jun over-expression. In addition to these effects, CEFs expressing Jun secrete factors that stimulate the motility of a human tongue carcinoma cell line. Our results suggest that Jun plays an important role in the potentiation of cell motility and invasion through multiple mechanisms. Int.

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat-1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I-EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src-transformed cells did not appear to be due to secreted transforming growth factor-alpha (TGF-alpha), since such factors were undetectable in culture fluids from the src-transformed cells. By several criteria of transformation, an EGF-receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down-modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.

We have previously shown that an intracellular mechanism down regulates epidermal growth factor (EGF) receptor levels in rodent fibroblasts transformed by the src oncogene (W. J. Wasilenko, L. K. Shawver, and M. J. Weber, J. Cell. Physiol. 131:450-457, 1987). We now report that this down regulation is due to an inhibition of EGF receptor biosynthesis. With Rat-1 (RI) cells infected with a temperature-sensitive src mutant, we found that 125I-labeled EGF binding to cells began to decrease soon after the activation of pp60v-sr by shift down to the permissive temperature for transformation. This effect of src on EGF receptors was reversible. Pulse-chase studies with [35SJmethionine-labeled cells revealed that the tyrosine protein kinase activity of pp6Ov-src had little if any effect on EGF receptor degradation rate. By contrast, the expression of pp6Ov-src caused a large reduction in the apparent rate of EGF receptor biosynthesis. Northern (RNA) blot analysis demonstrated that pp6Ov-src also caused marked reductions in the steady-state level of EGF receptor mRNA. These data indicate that one way the expression of the src oncogene can affect the machinery of growth control is by affecting the expression of specific genes for growth factor receptors.Oncogenes transform cells by virtue of their ability to mimic or interact with components of the machinery of normal growth regulation (10,11,22). Among the most thoroughly studied oncogenic agents is the src oncogene of Rous sarcoma virus, which encodes pp60vsrc, a 60-kilodalton tyrosine-specific protein kinase that associates with the inner surface of cellular membranes (8,21,34,35). Both the tyrosine protein kinase activity and the membrane association of this protein are required for transformation, but little else is known about the mechanism(s) by which pp60v-src disrupts normal cellular proliferation. In particular, it is unclear which step(s) in the control of normal growth and metabolism is directly altered by pp60vsrc. As a starting point for the investigation of this problem, we have examined the effects of pp60vsrc on the epidermal growth factor (EGF) receptor, since this well-characterized receptor is also a tyrosyl protein kinase (42) and an important determinant of growth regulation in a variety of normal and malignant cells (5,6,23,27). Moreover, published reports from S. J. Parsons and co-workers indicate that overexpression of c-src potentiates the mitogenic effects of EGF (28). These findings raise the possibility of interactions between src and the EGF receptor in the control of cellular growth and metabolism.In earlier studies, we reported that EGF receptor levels were markedly down modulated in rodent cells transformed by pp6Ov-src (43). Although the basis for this down modulation was unclear, it appeared to be dependent on the tyrosine kinase activity of pp6Ovsrc. In addition, we found little (if any) role for secreted transforming growth factors (39) in this phenomenon, leading us to suggest that the mechanism for receptor loss was based largely on ...