Farnesylation of Ras is important for the interaction with phosphoinositide 3-kinase gamma.
The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha- and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110gamma. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110gamma in vitro and in Sf9 cells, whereas the interaction with p110alpha is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110gamma and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110gamma.
Rubio
I
I
Research Unit Molecular Cell Biology, Medical Faculty, Friedrich- Schiller-University, Jena, Germany.
Wittig
U
U
Meyer
C
C
Heinze
R
R
Kadereit
D
D
Waldmann
H
H
Downward
J
J
Wetzker
R
R
D000818:Animals; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011485:Protein Binding; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D018631:ras Proteins / Q000378:metabolism*
D020558:GTP Phosphohydrolases; D020690:GTPase-Activating Proteins; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras); D011993:Recombinant Fusion Proteins; D018631:ras Proteins