The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.
After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.
Chen
Z
Z
Departments of Biochemistry and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686, USA.
Otto
J C
JC
Bergo
M O
MO
Young
S G
SG
Casey
P J
PJ
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D008745:Methylation; D008870:Microtubules / Q000502:physiology*; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology; D011107:Polylysine / Q000502:physiology*; D018631:ras Proteins / Q000737:chemistry / Q000502:physiology*
D011107:Polylysine; D018631:ras Proteins; D017239:Paclitaxel