Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf
Martin O. Bergo, … , Patrick J. Casey, Stephen G. Young
Martin O. Bergo, … , Patrick J. Casey, Stephen G. Young
Published February 15, 2004
Citation Information: J Clin Invest. 2004;113(4):539-550. https://doi.org/10.1172/JCI18829.
View: Text | PDF
Article Cell biology

Inactivation of Icmt inhibits transformation by oncogenic K-Ras and B-Raf

Abstract

Isoprenylcysteine carboxyl methyltransferase (Icmt) methylates the carboxyl-terminal isoprenylcysteine of CAAX proteins (e.g., Ras and Rho proteins). In the case of the Ras proteins, carboxyl methylation is important for targeting of the proteins to the plasma membrane. We hypothesized that a knockout of Icmt would reduce the ability of cells to be transformed by K-Ras. Fibroblasts harboring a floxed Icmt allele and expressing activated K-Ras (K-Ras-Icmtflx/flx) were treated with Cre-adenovirus, producing K-Ras-IcmtΔ/Δ fibroblasts. Inactivation of Icmt inhibited cell growth and K-Ras–induced oncogenic transformation, both in soft agar assays and in a nude mice model. The inactivation of Icmt did not affect growth factor–stimulated phosphorylation of Erk1/2 or Akt1. However, levels of RhoA were greatly reduced as a consequence of accelerated protein turnover. In addition, there was a large Ras/Erk1/2-dependent increase in p21Cip1, which was probably a consequence of the reduced levels of RhoA. Deletion of p21Cip1 restored the ability of K-Ras-IcmtΔ/Δ fibroblasts to grow in soft agar. The effect of inactivating Icmt was not limited to the inhibition of K-Ras–induced transformation: inactivation of Icmt blocked transformation by an oncogenic form of B-Raf (V599E). These studies identify Icmt as a potential target for reducing the growth of K-Ras– and B-Raf–induced malignancies.

Authors

Martin O. Bergo, Bryant J. Gavino, Christine Hong, Anne P. Beigneux, Martin McMahon, Patrick J. Casey, Stephen G. Young

×

Figure 5

Options: View larger image (or click on image) Download as PowerPoint
Mislocalization and increased steady-state levels of K-Ras in Icmt-defic...
Mislocalization and increased steady-state levels of K-Ras in Icmt-deficient fibroblasts. (a) Confocal micrographs of spontaneously immortalized Icmt+/+ and Icmt–/– fibroblasts that had been transfected with a GFP–K-Ras fusion construct. (b) Distribution of Ras proteins in the membrane (P100) and cytosolic (S100) fractions of K-Ras-Icmtflx/flx fibroblasts and the derivative K-Ras-IcmtΔ/Δ fibroblasts. The Ras proteins were immunoprecipitated from 1,000 μg of the indicated cell fractions with a pan-Ras–specific antibody, and a Western blot was performed with a K-Ras–specific antibody. (c) GTP-bound Ras proteins in K-Ras-Icmtflx/flx fibroblasts and derivative K-Ras-IcmtΔ/Δ fibroblasts. GTP-bound Ras proteins were precipitated from 1 × 106 K-Ras-Icmtflx/flx and K-Ras-IcmtΔ/Δ cells with the Ras-binding domain of Raf (Ras Activation Kit; Upstate Biotechnology Inc.), and a Western blot was performed with a pan-Ras antibody. (d) Northern blot of total RNA from K-Ras-Icmtflx/flx and K-Ras-IcmtΔ/Δ fibroblasts, hybridized with a mouse Kras2 cDNA probe. That probe detects a longer mouse Kras2 transcript (upper panel) and a shorter human activated K-Ras transcript (middle panel). The blot was stripped and hybridized with a Gapdh cDNA probe (lower panel). IP, immunoprecipitation; NB, Northern blot.