TY - JOUR
T1 - Proteomic characterization of cytoskeletal and mitochondrial class III β-tubulin
AU - Cicchillitti, Lucia
AU - Penci, Roberta
AU - Di Michele, Michela
AU - Filippetti, Flavia
AU - Rotilio, Domenico
AU - Donati, Maria Benedetta
AU - Scambia, Giovanni
AU - Ferlini, Cristiano
PY - 2008
Y1 - 2008
N2 - Class III β-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.
AB - Class III β-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.
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U2 - 10.1158/1535-7163.MCT-07-2370
DO - 10.1158/1535-7163.MCT-07-2370
M3 - Article
C2 - 18645017
AN - SCOPUS:51049119252
VL - 7
SP - 2070
EP - 2079
JO - Molecular Cancer Therapeutics
JF - Molecular Cancer Therapeutics
SN - 1535-7163
IS - 7
ER -