Multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma are dominantly inherited cancer syndromes. All three syndromes are associated with mutations in RET, which encodes a receptor-like tyrosine kinase. The altered RET alleles were shown to be transforming genes in NIH 3T3 cells as a consequence of constitutive activation of the RET kinase. The MEN2A mutation resulted in RET dimerization at steady state, whereas the MEN2B mutation altered RET catalytic properties both quantitatively and qualitatively. Oncogenic conversion of RET in these neoplastic syndromes establishes germline transmission of dominant transforming genes in human cancer.

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.

The ubiquitin pathway has been implicated in the regulation of the abundance of proteins that control cell growth and proliferation. We have identified and characterized a novel human ubiquitin isopeptidase, UBPY, which both as a recombinant protein and upon immunoprecipitation from cell extracts is able to cleave linear or isopeptide-linked ubiquitin chains. UBPY accumulates upon growth stimulation of starved human fibroblasts, and its levels decrease in response to growth arrest induced by cell-cell contact. Inhibition of UBPY accumulation by antisense plasmid microinjection prevents fibroblasts from entering S-phase in response to serum stimulation. By increasing or decreasing the cellular abundance of UBPY or by overexpressing a catalytic site mutant, we detect substantial changes in the total pattern of protein ubiquitination, which correlate stringently with cell proliferation. Our results suggest that UBPY plays a role in regulating the overall function of the ubiquitin-proteasome pathway. Affecting the function of a specific UBP in vivo could provide novel tools for controlling mammalian cell proliferation.

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, ϳ30% 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, ϳ3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (ϳ5%) 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-␥, 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.

Using a polymerase chain reaction approach, we have analyzed the alternative usage of the platelet-derived growth factor A-chain exon 6 in mRNA from various cell types. The results show that this sequence is utilized in a small fraction of the mRNA molecules in normal as well as transformed cells and that this phenomenon is conserved among mammalian species.