Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance.
Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.
Bhattacharya
Rajat
R
Department of Integrative Biology and Pharmacology, The University of Texas Medical School, Houston, TX 77030, USA.
Cabral
Fernando
F
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005455:Fluorescent Antibody Technique; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017239:Paclitaxel / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology*
D014404:Tubulin; D050257:Tubulin Modulators; D017239:Paclitaxel