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.