Anti-idiotypic antibodies that react with microtubule-associated proteins are present in the sera of rabbits immunized with synthetic peptides from tubulin's regulatory domain.

Abstract

A fundamental question in microtubule research is how the interactions of tubulin subunits with microtubule-associated proteins (MAPs) are controlled. The answer should provide insight into the regulation of the cellular processes in which microtubules are implicated. Previous work demonstrated the interaction ofMAPs with a 4-kDa C-terminal domain of tubulin a and 13 subunits. Synthetic peptides from the variable region of the 4-kDa C-terminal moiety of tubulin subunits, a-(430-441) and 13-(422-434), bind to MAP-2 and to the MAP tau, and a preferential interaction of the 13 peptide is observed. To define the regulatory significance of the substructure of the C-terminal tubulin domain, we produced rabbit antisera against these MAP-interacting peptides. We found that these antisera contained not only antibodies to the original synthetic peptides but also antibodies to MAPs. Here, we report that these antibodies, which react with MAP-1, MAP-2, and tau, appear to be a population of anti-idiotypic antibodies directed to the anti-peptide antibodies. They can inhibit MAPinduced tubulin assembly into microtubules in vitro, and the addition of MAPs overcomes the inhibition. The recognition by these anti-idiotypic antibodies of the tubulin-binding domain on MAPs provides unequivocal evidence that the tubulin region defined by the synthetic peptides is directly involved in the interaction with MAPs.The functional versatility of microtubules, in addition to their cellular dynamics, indicates the existence of fine regulatory signals modulating their assembly and intracellular orientation (1). The C-terminal moiety of tubulin plays a role in modulating the interactions responsible for tubulin selfassociation into microtubules (2, 3). Limited proteolysis of tubulin with subtilisin (between Glu-417 and Phe-418 in the a subunit; between Glu-407 and Phe-408 in the (3 subunit) produces the cleaved heterodimer and two 4-kDa peptide fragments containing the C-terminal domains of the a and (3 subunits, rendering tubulin assembly independent of microtubule-associated proteins (MAPs) (4). This 4-kDa domain appears to contain an essential site for MAP interactions (5, 6). Studies with exopeptidases that remove the last eight amino acid residues from tubulin subunits (7) and with antibodies that recognize the C-terminal tyrosine ofa-tubulin (8) indicate that the acidic, -1-kDa C-terminal subdomain of tubulin subunits is not directly involved in the binding of MAPs. Two synthetic peptides from the variable subdomain within the 4-kDa moiety of tubulin subunits were used in our studies, a-(430-441) (Lys-Asp-Tyr-Glu-Glu-Val-Gly-ValAsp-Ser-Val-Glu) and (3-(422-434) (Tyr-Gln-Gln-Tyr-GlnAsp-Ala-Thr-Ala-Asp-Glu-Gln-Gly). peptide exhibited a relatively strong interaction with two purified MAPs, MAP-2 and tau, whereas a-(430-441) interacted with these proteins with a rather low affinity, suggesting a differential capacity of the two tubulin subunits to bind selectively to MAPs (7). We report here the presence of MAP-reacting anti-idiotypi...

Structure of the bovine tau gene: alternatively spliced transcripts generate a protein family.

“…Each of the proposed acidic N-terminal, basic middle, and microtubule-binding C-terminal domains ( (27). Anti-idiotype antibodies isolated from sera after immunization with these peptides also bound to tau and MAP2 and interfered with their ability to promote microtubule assembly in vitro (34). This result suggests that MAP2 might also contain similar microtubulebinding domains which may have evolved by shuffling of exons encoding commonly used functional domains, as was suggested for many other proteins (18,40).…”

α‐, β‐, and γ‐tubulins: Sequence comparisons and structural constraints

“…The current analysis has therefore excluded this peptide (Fig. l), although it includes the MAP2/tau binding site [e.g., Rivas et al, 1988;Vera et al, 19891 and a neuronal P,-tubulin isotype-specific phosphorylation site [Diaz-Nido et al, 19901. Structure prediction has indicated that the C-terminal peptide (1x414-447 and P:403-443) is folded to form a coiled coil [Lupus et a]., 19911: the marked heterogeneity from ~4 4 1 and P:431 suggests that the prob- (A) The amino acid sequence, in single letter code, of mouse a l , chick PI, and Aspergillus nidulans y-tubulin.…”

Functional expression of murine multidrug resistance in Xenopus laevis oocytes.

“…The 20,000 x g pellet was resuspended in electrophoresis sample buffer (32) and fractionated by SDS/polyacrylamide gel electrophoresis (32). Proteins were transferred electrophoretically to nitrocellulose membranes (33) and probed with the antipeptide antibody mdr (Ab-1) (Oncogene Science, Manhasset, NY). This is a rabbit, affinity-purified polyclonal antibody raised against the peptide Cys-Ala-Leu-Asp-Thr-GluSer-Glu-Lys-Val-Val-Gln-Glu-Ala-Leu-Asp-Lys-Ala-ArgGlu-Gly from the C-terminal region of P glycoprotein.…”