Activation and Nuclear Translocation of Mitogen-activated Protein Kinases by Polyomavirus Middle-T or Serum Depend on Phosphatidylinositol 3-Kinase

Abstract

Several cellular signal transduction pathways activated by middle-T in polyomavirus-transformed cells are required for viral oncogenicity. Here we focus on the role of phosphatidylinositol 3-kinase (PI 3-kinase) and Ras and address the question how these signaling molecules cooperate during cell cycle activation. Ras activation is mediated through association with SHC.GRB2.SOS and leads to increased activity of several members of the mitogen-activated protein (MAP) kinase family, while activation of PI 3-kinase results in the generation of D3-phosphorylated phosphatidylinositides whose downstream targets remain elusive. PI 3-kinase activation might also ensue as a direct consequence of Ras activation. Oncogenicity of middle-T requires stimulation of both Ras- and PI 3-kinase-dependent pathways. Mutants of middle-T incapable to bind either SHC.GRB2.SOS or PI 3-kinase are not oncogenic. Sustained activation and nuclear localization of one of the MAP kinases, ERK1, was observed in wild type but not in mutant middle-T-expressing cells. Wortmannin, an inhibitor of PI 3-kinase, prevented MAP kinase activation and nuclear localization in middle-T-transformed cells. PI 3-kinase activity was also required for activation of the MAP kinase pathway in normal serum-stimulated cells, generalizing the concept that signaling through MAP kinases requires not only Ras-but also PI 3-kinase-mediated signals.

Mapping of polyomavirus middle T domain that is responsible for AP-1 activation

“…This result points to an essential role for PI3K in the AP-1 activation by MT. Urich et al (1995) have shown that MT activates and induces MAPK nuclear translocation, this e ect being dependent upon MT binding to Shc and PI3K. In our experiments, MT binding to Shc and PI3K also seems to be essential for complete MAPK activation (data not shown).…”

“…However, high AP-1 activity is observed in the 250phe mutant (that does not bind Shc) even though the Erk1 activity displayed by this cell line is low. Interestingly, the 250phe mutant is only 50% less e ective than WTMT to induce the uPA promoter (Urich et al, 1995). As discussed by the authors (Urich et al, 1995) low levels of MAPK in the nucleus, undetectable by immunostaining, could be present in the 250phe cell line.…”

“…Particularly, MAPK activation has been shown to be associated with AP-1 activation in other systems (Marra et al, 1995;Urich et al, 1997). Urich et al (1995) have shown that nuclear translocation of MAPKs by MT or serum is dependent on PI3K activity.…”

“…The use of wortmannin as a potent and speci®c inhibitor of PI3K (Powis et al, 1994) was shown to block both MAPK translocation to the nucleus in MT overexpressing and in serum stimulated cells (Urich et al, 1995) and activation of pp70 s6k in WT and 250phe mutants (Dahl et al, 1996). To test whether inactivation of PI3K by this speci®c inhibitor would alter the AP-1 activity displayed by wild type MT cell line, we performed gel shift assays using lysates from cultures treated with 100 nM wortmannin for di erent periods of time (data not shown).…”

“…Generation of a large number of MT mutants (Markland and Smith, 1987;Druker and Roberts, 1991;Carmichael et al, 1984;Morgan et al, 1988; has been useful in identi®cation of the MT domains that are responsible for binding to these cellular proteins as well as to the analysis of their importance in MT-induced cell transformation (Glenn and Eckhart, 1993;Campbell et al, 1994Campbell et al, , 1995Urich et al, 1995;Dahl et al, 1996).…”