Mutations Affecting β-Tubulin Folding and Degradation

Abstract

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) ␤-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable ␤-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective ␤-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with ␤-tubulin in purified ␣␤ heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded ␤-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of ␤-tubulin from the mutant allele causes a 30 -40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.Microtubules are essential cytoskeletal structures responsible for much of the vesicular traffic in cells, organization of the endoplasmic reticulum and Golgi apparatus, and segregation of sister chromatids before cell division. They assemble by the endwise concatenation of ␣␤ tubulin heterodimers to form protofilaments, 13 of which associate laterally to form the walls of microtubules in most eukaryotic cells. ␣-and ␤-tubulin subunits each follow a complex folding pathway before joining to form assembly-competent heterodimers. This process involves prefoldin (1, 2), CCT 2 (cytosolic chaperonin containing TCP-1, also called TCP-1 complexes, TriC, or cytosolic chaperonin) (3-5), and the downstream participation of a series of protein cofactors (6 -9). Prefoldin binds to nascent chains and is thought to target newly synthesized tubulin to CCT (1), which then releases these polypeptides in a quasinative state. The released ␤-tubulin is captured by either cofactor A or cofactor D. In a parallel pathway, quasi-native ␣-tubulin polypeptides are captured by either cofactor B or cofactor E. Cofactor E/␣ and cofactor D/␤ complexes interact to form a supercomplex. Entry of cofactor C into this supercomplex triggers GTP hydrolysis by ␤-tubulin, a reaction that results in the release of newly formed assembly-competent ␣␤ tubulin heterodimers (6, 7).Mammalian cells express multiple tubulin genes, each of which encodes a distinct ␣-or ␤-tubulin isotype (10, 11). As far as is known, these distinct gene products follow a common folding pathway. In addition to their role in the de novo assembly of tubulin dimers, cofactors C, D, and E act in concert on native heterodimers as GTPase-activating proteins. This reaction occurs at a tubulin concentration far below that required for polymerization into microtubules and is thought to act primarily as a quality control mechanism...

N-(4-iodophenyl)-N′-(2-chloroethyl)urea as a microtubule disrupter: in vitro and in vivo profiling of antitumoral activity on CT-26 murine colon carcinoma cell line cultured and grafted to mice

“…That phenomenon is not unique, as it has been already reported for other antimicrotubule agents by Schimming et al (1999). We hypothesise that cells with alkylated b-tubulin might be eliminated quickly in vivo via phagocytosis and/or ubiquitin proteasome degradation pathways, as described previously (Krysko et al, 2006;Liu et al, 2006;Wang et al, 2006). Likewise, the tumoricidal concentration of ICEU necessary to kill cells should be higher as compared to in vitro when one considers the interrelated influence of physiological parameters such as perfusion, necrosis, cell density and interstitial pressure that may affect the diffusion of the drug into the tumoral tissues and the neoplastic cells.…”

Large spectrum of lissencephaly and pachygyria phenotypes resulting from de novo missense mutations in tubulin alpha 1A (TUBA1A)

“…It has recently been shown that beta-tubulin with a p.L187R mu11tation, fails to form heterodimers, and consequently does not incorporate into MTs. This was shown to be a consequence of an impaired interaction with cofactors involved in the tubulin folding pathway [Wang et al, 2006]. The same molecular impairment may underlie the pathology of the individual with the I188L mutation.…”

Global Regulation of the Interphase Microtubule System by Abundantly Expressed Op18/Stathmin

“…Note that the relevance of this mechanism for regulated mRNA stability under steady-state conditions and for specifying cellular tubulin content is still unclear. Indeed, extensive studies of selected variants of CHO cells with acquired resistance to taxol have provided examples of cell lines in which the taxol/colchicine-responsive autoregulatory mechanism does not compensate for alterations in tubulin heterodimer content and/or partitioning (Boggs and Cabral, 1987;Barlow et al, 2002;Wang et al, 2006). Moreover, it has also been shown that increased transcription is the major mechanism behind increased tubulin heterodimer content during pressure-induced hypertrophy of cardiac cells, and changes in mRNA stability could not be detected (Narishige et al, 1999).…”

“…However, detailed analyses of cell lines with acquired resistance to the microtubule-stabilizing drug taxol have shown that substantial changes in tubulin heterodimer content, monomer-polymer partitioning, or both may occur without compensatory changes in tubulin synthesis (Boggs and Cabral, 1987;Barlow et al, 2002). Thus, the significance of autoregulation of tubulin mRNA stability for the normal regulation of tubulin heterodimer content and partitioning is still unclear, and it has been proposed that autoregulation is not activated by small changes in polymerization (Wang et al, 2006).…”