Activation of H-ras61L-specific signaling pathways does not require posttranslational processing of H-ras.
We have previously demonstrated that H-ras61L retained transforming activity when lacking C-terminal lipid modifications, provided that plasma membrane localization was restored by an N-terminal transmembrane domain. Since several ras-activated pathways contribute to the transformed phenotype, we utilized a novel set of transmembrane domain-anchored H-ras derivatives to examine if lipids are required for activation of any specific signaling pathways. We demonstrate here that H-ras61L-induced activation of the Raf/MEK/MAPK pathway, including recruitment of Raf to the plasma membrane and activation of Raf and MAPK, does not require C-terminal processing of H-ras61L. Biochemical fractionation experiments confirm the localization of TM-ras derivatives to the plasma membrane, as well as the ras-mediated recruitment of c-Raf-1. Changes in the actin cytoskeleton, controlled by H-ras61L-mediated activation of the Rac/ Rho pathway, as well as PI 3-kinase activation, can also occur in the absence of C-terminal lipid modifications. Finally, downstream events, such as the induction of the immediate-early gene c-fos or neurite outgrowth in PC12 cells, are stimulated by the expression of plasma membrane-anchored, nonlipidated H-ras6lL. These results demonstrate that H-ras can be functionally targeted to the plasma membrane using a transmembrane domain sequence and that several signal transduction pathways downstream of H-ras can be activated without the presence of normal lipid modifications.
Hart
K C
KC
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla 92093-0367, USA.
Robertson
S C
SC
Donoghue
D J
DJ
D000818:Animals; D002454:Cell Differentiation; D020935:MAP Kinase Signaling System; D016716:PC12 Cells / Q000166:cytology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
D018631:ras Proteins