Intra-allelic suppression of a mutation that stabilizes microtubules and confers resistance to colcemid.
Cmd 4 is a colcemid resistant beta-tubulin mutant of Chinese hamster ovary cells that exhibits hypersensitivity to paclitaxel and temperature sensitivity for growth. The mutant beta-tubulin allele in this cell line encodes a D45Y amino acid substitution that produces colcemid resistance by making microtubules more stable. By selecting revertants of the temperature sensitive and paclitaxel hypersensitive phenotypes, we have identified three cis-acting suppressors of D45Y. One suppressor, V60A, maps to the same region as the D45Y alteration, and a second suppressor, Q292H, maps to a distant location. Both appear to produce compensatory changes in microtubule assembly that counteract the effects of the original D45Y substitution. Consistent with this view, expression of the V60A mutation in transfected wild-type cells produced paclitaxel resistance and greatly decreased microtubule assembly. Additionally, it produced a paclitaxel-dependent phenotype in which cells grew normally in the presence, but not the absence, of the drug. The Q292H mutation caused even greater disassembly of microtubules such that cells were unable to proliferate when the transgene was expressed; but, unlike the V60A mutation, cell growth could not be rescued by paclitaxel. A third suppressor, A254V, maps to a region near the interface between alpha- and beta-tubulin that contains the colchicine binding site. Although it made transfected wild-type cells hypersensitive to colcemid, it did not affect paclitaxel or vinblastine sensitivity, nor did it reduce microtubule assembly. We suggest that this mutation acts by increasing tubulin's affinity for colcemid.
Wang
Yaqing
Y
Department of Integrative Biology and Pharmacology, University of Texas Medical School, 6431 Fannin Street, Houston, Texas 77225, USA.
Veeraraghavan
Sudha
S
Cabral
Fernando
F
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D003703:Demecolcine / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D008870:Microtubules / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017239:Paclitaxel / Q000494:pharmacology; D014162:Transfection; D014404:Tubulin / Q000235:genetics; D014747:Vinblastine / Q000494:pharmacology
D000972:Antineoplastic Agents, Phytogenic; D014404:Tubulin; D014747:Vinblastine; D017239:Paclitaxel; D003703:Demecolcine