Prenylation-dependent association of Ki-Ras with microtubules. Evidence for a role in subcellular trafficking.
We recently identified a prenyl peptide-binding protein in microsomal membranes from bovine brain (Thissen, J. A., and Casey, P. J. (1993) J. Biol. Chem. 268, 13780-13783). Through a variety of approaches, this binding protein has been identified as the cytoskeletal protein tubulin. Prenyl peptides bind to purified tubulin with a Kd of 40 nM and also bind to tubulin polymerized into microtubules. Microtubule affinity chromatography of extracts from cells in which the prenyl protein pool was metabolically labeled revealed that prenyl proteins bound to the immobilized microtubules; one, a 24-kDa protein, was tentatively identified as a GTP-binding protein. Of several prenylated GTP-binding proteins tested, including Ki-Ras4B, Ha-Ras, RhoB, RhoA, and Rap1B, only Ki-Ras was found to bind significantly to microtubules, and this was in a prenylation-dependent fashion. A potential significance of the interaction of Ki-Ras4B with microtubules was indicated from analysis of the localization of newly synthesized Ki-Ras4B and Ha-Ras, each tagged with green fluorescence protein (GFP). Treatment of NIH-3T3 cells expressing GFP-Ki-Ras with Taxol (paclitaxel) resulted in accumulation of the expressed protein in intracellular locations, whereas in control cells the protein was correctly targeted to the plasma membrane. Importantly, such treatment with paclitaxel did not affect the cellular localization of expressed GFP-Ha-Ras. These results indicate that an intact microtubule network may be directly involved in Ki-Ras processing and/or targeting and provide direct evidence for a physiological distinction between Ki-Ras and Ha-Ras in cells. Additionally, the finding that paclitaxel treatment of cells disrupts Ki-Ras trafficking suggests an additional mechanism for the anti-proliferative effects of this drug.
Thissen
J A
JA
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710-3686, USA.
Gross
J M
JM
Subramanian
K
K
Meyer
T
T
Casey
P J
PJ
D000818:Animals; D001921:Brain; D002417:Cattle; D002451:Cell Compartmentation; D002846:Chromatography, Affinity; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D014404:Tubulin; D016283:Proto-Oncogene Proteins p21(ras); D017239:Paclitaxel