Targeted Inactivation of the Isoprenylcysteine Carboxyl Methyltransferase Gene Causes Mislocalization of K-Ras in Mammalian Cells

Martin O Bergo,Gordon K Leung,Patricia Ambroziak,James C Otto,Patrick J Casey,Stephen G Young

doi:10.1074/jbc.c000079200

Martin O Bergo, Gordon K Leung + Show 4 more

Open Access

https://doi.org/10.1074/jbc.c000079200

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Abstract

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.

Highlights

Ras proteins, as well as other proteins that terminate with a so-called CaaX sequence, undergo several sequential posttranslational processing steps
To produce Icmt2/2 cells, two Icmt1/2 clones were subjected to selection in high concentrations of G418
Icmt2/2 cell densities were reduced by 5–30% at 24, 48, and 72 h, compared with the Icmt1/1 cells (p, 0.01, data not shown)

Summary

Introduction

As well as other proteins that terminate with a so-called CaaX sequence, undergo several sequential posttranslational processing steps. One potential means of approaching this issue would be to competitively inhibit the isoprenylcysteine carboxyl methyltransferase with small, cell-permeable, methyl-accepting isoprenylated substrates (e.g., N-acetyl-Sfarnesyl-L-cysteine or N-acetyl-S-geranylgeranyl-L-cysteine) [13,14,15,16]. The drawback of this approach, is that these. The fact that these molecules contain isoprenyl groups could lead them to displace isoprenylated proteins from their cellular binding sites [17] To avoid such problems, we used genetargeting techniques to produce a mammalian cell line lacking isoprenylcysteine carboxyl methyltransferase. We document the production of a mouse cell line that is homozygous for an Icmt knockout mutation, and we demonstrate that K-Ras localization is abnormal in those cells

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