Increased Levels of a Unique Post-translationally Modified βIVb-Tubulin Isotype in Liver Cancer

Microtubules are dynamic protein polymers that play an essential role in cell division, maintenance of cell shape, transport of vesicles and cell motility (1). These hollow cylinders are constructed of heterodimers of α- and β-tubulin, which bind head-to-tail to form protofilaments that associate laterally to form microtubules (2). There are 6 α- and 7 β-tubulin genes described in mammals, with each gene being highly conserved across species (3). The majority of the diversity in tubulin gene products, termed isotypes, occurs in the last 15–20 residues of the C-terminus, which is known as the isotype defining region and is located on the outer surface of microtubules (4). Further increasing the diversity of the tubulin isotypes is their ability to undergo a range of post-translational modifications. Common post-translational modifications to tubulin are polyglutamylation, tyrosination/detyrosination, polyglycylation, acetylation and phosphorylation, most of which occur in the C-terminal region (5). Although there is no consensus regarding the specific role or function of individual tubulin isotypes, tissue-specific changes in the expression of tubulin isotypes have been observed, and isotype composition has been demonstrated to affect the dynamics of microtubule assembly (6–8). It has been suggested that divergent C-termini may provide a mechanism for isotype-specific microtubule associated protein (MAP) binding (9). Recently, a tubulin code, similar to the histone code, was proposed whereby tubulin isotypes and their modifications could serve as discrete signals to modulate cellular events, suggesting a mechanistic role for the observed diversity in microtubules (10). The integrity of the cytoskeleton is essential for the proper functioning of all cells. Therefore, changes in the composition of microtubules, a major component of the cytoskeleton, could contribute to tumorigenesis. Previous studies have examined the α- and β-tubulin isotypes in both tumors and cancer cell lines. An increase in βIII tubulin is the most commonly described alteration in tubulin expression in cancer, with an increase of this neuronal isotype observed in a variety of tumors (11–15). An increase in the expression of the βII tubulin in cancer has also been described (16). While a majority of studies have focused on β-tubulins, a recent report demonstrated a shift in post-translational modifications of α-tubulin isotypes in prostate cancer cell lines (17). In these studies, isotypes were assigned either at the protein level using isotype-specific antibodies or at the mRNA level by RT-PCR. Both of these methods continue to provide important qualitative and quantitative data about tubulin isotypes. However, neither method is able to completely describe the tubulin isotypes at the protein level. Mass spectrometry (MS) complements traditional methods of characterizing tubulin isotypes (18). It is an ideal tool for the identification of post-translational modifications to tubulins, with the ability to characterize multiple modifications in one experiment (19, 20). Unlike experiments employing antibodies, mass spectrometry can be used to detect mutations in isotypes as well as to identify new isotypes (21, 22). Also, when combined with “stable isotope labeling with amino acids in culture” (SILAC), mass spectrometry provides information regarding the relative expression levels of tubulin isotypes in drug resistant cell lines as compared to the tubulin isotypes present in drug sensitive cell lines (23). Therefore, the application of mass spectrometry to tubulin isotype characterization can provide unique information about tubulin isotypes at the protein level that cannot readily be achieved with other methods. Using a highly reproducible rat liver cancer model, the resistant hepatocyte model, we profiled the tubulin isotypes present at early, middle and late stages of cancer development (24). Hepatocellular carcinoma (HCC) is one of the most common and fatal forms of cancer worldwide (25). Detection of HCC at early stages is difficult and treatment options after identification are limited and often ineffective (26). In order to gain a better understanding of the changes occurring at the molecular level in carcinogenesis, we employed high resolution isoelectric focusing along with cyanogen bromide (CNBr) cleavage and MS to identify the tubulin isotypes present in the liver. Our methodology allowed for the identification of a novel C-terminal modification to βIVb-tubulin. We quantitated the relative amount of the peptide harboring this novel post-translational modification compared to all β-tubulin isotypes, and when comparing the levels in rat liver which underwent chemical carcinogen treatment relative to control liver, we demonstrated a 3-fold increase in modification during later stages of cancer. Such changes in a post-translational modification of a tubulin isotype during progression of liver cancer suggest that alterations to microtubules may be a useful indicator for the detection and treatment of liver cancer.