Overexpression of p97^Eps8 leads to cellular transformation: implication of pleckstrin homology domain in p97^Eps8-mediated ERK activation

Abstract

Two isoforms of Eps8, p97^Eps8 and p68^Eps8, have been identified as the substrates for receptor tyrosine kinases. Our previous studies indicated that both tyrosyl phosphorylation and protein expression of Eps8 were elevated in v-Src transformed cells. In an attempt to examine the role played by p97^Eps8 in tumorigenesis, we have first obtained cells overexpressing p97^Eps8 and its pleckstrin homology (PH)-truncated variant. We then demonstrated that cells overexpressing p97^Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. Furthermore, elevated serum-induced extracellular responsive kinase (ERK) activation was observed in p97^Eps8 overexpressors. This enhanced ERK activation was sensitive to a MEK1 specific inhibitor PD98059 and was important for p97^Eps8-mediated transformation, since transfection of vectors expressing dominant negative MEK1 and p97^Eps8 abrogated focus formation by p97^Eps8. In contrast, PH-truncated p97^Eps8 failed to localize at the plasma membrane and that the truncated variant also did not elevate ERK activation and cellular transformation in response to serum stimulation. Our results thus indicated that: (i) the gene encoding p97^Eps8 was an oncogene; (ii) p97^Eps8-induced oncogenesis was partly mediated by ERK activation; and (iii) the PH domain of p97^Eps8 was critical for its cellular localization, ERK activation and its ability to transform cells.