Proteomic characterization of cytoskeletal and mitochondrial class III beta-tubulin.
Class III beta-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function.
Cicchillitti
Lucia
L
Department of Oncology, Catholic University of the Sacred Heart, Largo A. Gemelli, 1-86100, Campobasso, Italy.
Penci
Roberta
R
Di Michele
Michela
M
Filippetti
Flavia
F
Rotilio
Domenico
D
Donati
Maria Benedetta
MB
Scambia
Giovanni
G
Ferlini
Cristiano
C
D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D000730:Androstadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004586:Electrophoresis; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics; D014404:Tubulin / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D020033:Protein Isoforms; D014404:Tubulin; D014415:Tunicamycin; D000077191:Wortmannin