Involvement of phosphatidylinositol 3-kinase, but not RalGDS, in TC21/R-Ras2-mediated transformation.
Oncogenic Ras and activated forms of the Ras-related protein TC21/R-Ras2 share similar abilities to alter cell proliferation. However, in contrast to Ras, we found previously that TC21 fails to activate the Raf-1 serine/threonine kinase. Thus, TC21 must utilize non-Raf effectors to regulate cell function. In this study, we determined that TC21 interacts strongly with some (RalGDS, RGL, RGL2/Rlf, AF6, and the phosphatidylinositol 3-kinase (PI3K) catalytic subunit p110delta), and weakly with other Ras small middle dotGTP-binding proteins. In addition, library screening identified novel TC21-interacting proteins. We also determined that TC21, similar to Ras, mediates activation of phospholipase Cepsilon. We then examined if RalGDS, a RalA guanine nucleotide exchange factor, or PI3K are effectors for TC21-mediated signaling and cell proliferation in murine fibroblasts. We found that overexpression of full-length RalGDS reduced the focus forming activity of activated TC21. Furthermore, expression of activated Ras, but not TC21, enhanced GTP loading on RalA. In fact, TC21 attenuated insulin-stimulated RalA small middle dotGTP formation. In contrast, like Ras, expression of activated TC21 resulted in membrane translocation and an increase in the PI3K-dependent phosphorylation of Akt, and inhibition of PI3K activity interfered with TC21 focus formation. Finally, unlike Ras, TC21 did not activate the Rac small GTPase, indicating that Ras may not activate Rac by PI3K. Taken together, these results suggest that PI3K, but not RalGDS, is an important mediator of cell proliferation by TC21.
Murphy
Gretchen A
GA
Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA. gmurphy@med.unc.edu
Graham
Suzanne M
SM
Morita
Staeci
S
Reks
Sarah E
SE
Rogers-Graham
Kelley
K
Vojtek
Anne
A
Kelley
Grant G
GG
Der
Channing J
CJ
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015723:Gene Library; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D020559:Monomeric GTP-Binding Proteins; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D020798:Two-Hybrid System Techniques; D010738:Type C Phospholipases / Q000378:metabolism; D020725:ral GTP-Binding Proteins; D020726:ral Guanine Nucleotide Exchange Factor / Q000378:metabolism*
D002867:Chromones; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009025:Morpholines; D011993:Recombinant Fusion Proteins; D020726:ral Guanine Nucleotide Exchange Factor; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon; C490200:Rras2 protein, mouse; D020558:GTP Phosphohydrolases; C492198:RALA protein, human; C118120:Rala protein, mouse; D020559:Monomeric GTP-Binding Proteins; D020725:ral GTP-Binding Proteins