Israr A. Khan

Tubulin structure and biochemistry.

Abstract In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands — both microtubule-associated proteins and anti-mitotic drugs — are becoming clearer at the molecular level.

THE TUMOR SUPPRESSOR PROTEIN FHIT : A NOVEL INTERACTION WITH TUBULIN

FHIT (fragilehistidine triad) is a candidate human tumor suppressor gene located at chromosome 3p14.2, a location that encompasses the FRA3B chromosomal fragile site. Aberrant transcripts have been detected in a variety of primary tumors, and homozygous deletions in the FHIT locus have been detected in different tumor cell lines. The gene product Fhit in vitro possesses the ability to hydrolyze diadenosine 5′,5′′′-P1,P3-triphosphate (Ap3A). The mechanism of action of Fhit as a tumor suppressor is unknown. Because the tubulin-microtubule system plays an important role in cell division and cell proliferation, we investigated the interaction between wild-type Fhit or mutant Fhit (H96N) and tubulin in vitro. The mutant form of Fhit (H96N) lacks Ap3A hydrolase activity but retains tumor suppressor activity. We found that both wild-type and mutated forms of Fhit bind to tubulin strongly and specifically with K d values of 1.4 and 2.1 μm, respectively. Neither wild-type nor mutant Fhit cause nucleation or formation of microtubules, but in the presence of microtubule-associated proteins, both wild-type and mutant Fhit promote assembly to a greater extent than do microtubule-associated proteins alone, and the microtubules formed appear normal by electron microscopy. Our results suggest the possibility that Fhit may exert its tumor suppressor activity by interacting with microtubules and also indicate that the interaction between Fhit and tubulin is not related to the Ap3A hydrolase activity of Fhit.

POSSIBLE REGULATION OF THE IN VITRO ASSEMBLY OF BOVINE BRAIN TUBULIN BY THE BOVINE THIOREDOXIN SYSTEM

Microtubule assembly in vitro and in vivo is highly sensitive to a variety of sulfhydryl-reactive reagents, raising the question of the possible existence of a physiological sulfhydryl-mediated system for regulating microtubule assembly. However, the specific reagents which have previously been used to inhibit microtubule assembly in vitro are either nonphysiological or, if physiological, effective only at concentrations much higher than their physiological ones. Because of reports of association in vivo between microtubules and the sulfhydryl-reactive proteins thioredoxin and thioredoxin reductase, we decided to examine the interaction in vitro between microtubules and the thioredoxin system, comprising thioredoxin, thioredoxin reductase and NADPH. At pH 6.8, both the mammalian and the Escherichia coli thioredoxin systems inhibited microtubule assembly by 4-35% (19 +/- 9%) by reducing one intra-subunit disulfide bond in the tubulin dimer. The thioredoxin-reducible disulfide of the tubulin dimer remains protected from thioredoxin in the assembled microtubules. Thioredoxin or thioredoxin reductase alone, or together in the absence of NADPH, were incapable of either reducing tubulin or inhibiting microtubule assembly. Microtubules formed from reduced tubulin were found to be stable and morphologically identical to those obtained from native tubulin dimers. Since the components of the thioredoxin system were used at concentrations similar to their physiological ones, our results suggest a potential role of the thioredoxin system in regulation of microtubule assembly in vivo.

Different Effects of Vinblastine on the Polymerization of Isotypically Purified Tubulins from Bovine Brain

Vinblastine, a highly successful antitumor drug, targets the tubulin molecule. Tubulin, the subunit protein of microtubules, consists of an α- and a β-subunit, both of which consist of isotypes encoded by different genes. We have purified three isotypes of bovine brain tubulin, namely, αβII, αβIII and αβIV. Microtubule associated protein-2 (MAP2) and Tau-induced assembly of these isotypes were compared in the presence and absence of vinblastine. MAP2-induced assembly of unfractionated tubulin and all the isotypes except αβII tubulin was resistant to 1 μM vinblastine. Vinblastine at low concentrations (<10 μM) progressively inhibited the assembly of all of the isotypes but the vinblastine concentration required for inhibition of MAP2-induced microtubule assembly was minimal for αβII. The tau-induced assembly of unfractionated tubulin and αβIII were equally sensitive to 1 μM vinblastine whereas αβII and αβIV were much more sensitive to vinblastine. The microtubules obtained in the presence of tau from unfractionated tubulin, αβII and αβIV could be easily aggregated by 20 μM vinblastine whereas such as aggregation of microtubules obtained from αβIII and tau required approximatedly 40 μM vinblastine. Our results suggest that among the tubulin isotypes, αβII is the most sensitive to vinblastine in the presence of MAPs while αβIII is the most resistant and this intrinsic resistance of αβIII dimers persists in the polymeric form of αβIII tubulin as well. These results may be relevant to the therapeutic and toxic actions of vinblastine.

Tubulin structure and biochemistry

In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands--both microtubule-associated proteins and anti-mitotic drugs--are becoming clearer at the molecular level.