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

Two isoforms of Eps8, p(97Eps8) and p68(Eps8), have been identified as the substrates for receptor tyrosine kinases, Our previous studies indicated th at both tyrosyl phosphorylation and protein expression of Eps8 were elevate d in v-Src transformed cells, In an attempt to examine the role played by p 97(Eps8) in tumorigenesis, we have first obtained cells overexpressing p97( Eps8) and its pleckstrin homology (PH)-truncated variant. We then demonstra ted that cells overexpressing p97(Eps8) not only exhibited the ability of f ocus formation in cell culture but also promoted the tumor formation in mic e as compared to controls, Furthermore, elevated serum-induced extracellula r responsive kinase (ERK) activation was observed in p97(Eps8) overexpresso rs, This enhanced ERK activation was sensitive to a MEK1 specific inhibitor PD98059 and was important for p97(Eps8)-mediated transformation, since tra nsfection of vectors expressing dominant negative MEK1 and p97(Eps8) abroga ted 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 seru m stimulation, Our results thus indicated that: (1) the gene encoding p97(E ps8) was an oncogene; (ii) p97(Eps8)-induced oncogenesis was partly mediate d by ERK activation; and (iii) the PH domain of p97(Eps8) was critical for its cellular localization, ERK activation and its ability to transform cell s.