P P Di Fiore

eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

Efficient coupling with phosphatidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members.

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFR/ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10- to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase C gamma and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.

One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.

A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.

Different structural alterations upregulate in vitro tyrosine kinase activity and transforming potency of the erbB-2 gene.

Compared with normal erbB-2 gp185, mutant erbB-2 proteins generated by mutations either in the transmembrane domain or by NH2-terminal deletion are able to transform NIH 3T3 cells at a 10- to 100-fold greater efficiency. Mutant proteins of both classes show increased tyrosine kinase activity, suggesting that an abnormal level of receptor-associated tyrosine kinase activity is a major determinant of erbB-2 oncogenic potential.

Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals.

A method which allows direct cloning of intracellular substrates for receptor tyrosine kinases (RTKs) was developed. By applying this technique to the study of the epidermal growth factor receptor (EGFR) signaling pathway, we have isolated a cDNA, designated eps8, which predicts a approximately 92 kDa protein containing an SH3 domain. Eps8 also contains a putative nuclear targeting sequence. Antibodies specific to the eps8 gene product recognize a protein of M(r) 97 kDa and a minor 68 kDa component, which are closely related, as demonstrated by V8 proteolytic mapping. The product of the eps8 gene is tyrosine‐phosphorylated in vivo following EGF stimulation of intact cells and associates with the EGFR, despite the lack of a functional SH2 domain. Several other RTKs are also able to phosphorylate p97eps8. Thus, the eps8 gene product represents a novel substrate for RTKs. Adoptive expression of the eps8 cDNA in fibroblastic or hematopoietic target cells expressing the EGFR resulted in increased mitogenic response to EGF, implicating the eps8 gene product in the control of mitogenic signals.

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes.

Transformation of a rat thyroid epithelial cell line (FRTL5‐C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid‐soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC‐C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v‐mos oncogene) and when transformed by transfection with the c‐myc proto‐oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c‐myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI‐like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto‐oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non‐neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non‐neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprungs disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild‐type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972–>Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32–>Leu) and HSCR393 (Phe393–>Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway.

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

Breast cancer metastases are molecularly distinct from their primary tumors

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether ‘phenotypic’ disease progression is really associated with ‘molecular’ progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

Constitutive Phosphorylation of eps8 in Tumor Cell Lines: Relevance to Malignant Transformation

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy), corresponding to the 50% effective dose of EGF for mitogenesis, approximately 3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (approximately 5%) similar to that associated with potent mitogenic response in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and shc might be part of a pathway which is preferentially selected in some tumors. Cooperation between these two transducers was further indicated by the finding of their in vivo association. This association was, at least in part, dependent on recognition of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alterations can contribute to the malignant phenotype.