Tubulin Peptide Research Articles

Structure of the polyglutamyl side chain posttranslationally added to alpha-tubulin.

Polyglutamylation, a new posttranslational modification of tubulin identified originally on the acidic alpha variants by Eddé et al. (Eddé, B., Rossier, J., Le Caer, J. P., Desbruyeres, E., Gros, F., and Denoulet, P. (1990) Science 247, 83-85), consists of the successive addition of glutamyl units to the Glu445. To characterize their linkage mode mouse tubulin was posttranslationally labeled with [3H]glutamate. After digestion of [3H]tubulin with thermolysin, up to eight radioactive peaks were separated on an anion exchange column (DEAE). Combined use of Edman degradation sequencing and mass spectrometry analysis of the first 6 one indicated that they all correspond to the same COOH-terminal sequence 440VEGEGEEEGEE450 bearing one to six glutamyl units on the Glu445. The first glutamyl residue is amide-linked to the gamma-carboxyl group of Glu445, but the additional residues can be linked to the gamma- or alpha-carboxyl groups of the preceding one. All possible linkages for the biglutamylated tubulin peptides (gamma 1 alpha 2, gamma 1 gamma 2) and triglutamylated (gamma 1 alpha 2 alpha 3, gamma 1 alpha 2 gamma 3, gamma 1 alpha 2 gamma 2, gamma 1 gamma 2 alpha 3, gamma 1 gamma 2 gamma 3) were synthesized. These different peptides were successfully separated on a C18 5-micron reverse phase column. We found that the bi- and triglutamylated tubulin peptides behave as the gamma 1 alpha 2 and gamma 1 alpha 2 alpha 3 synthetic peptides, respectively. These results indicate that the second and third glutamyl residues of the polyglutamyl side chain are amide-linked to the alpha-carboxyl group of the preceding unit.

MAP-1 and MAP-2 binding sites at the C-terminus of .beta.-tubulin. Studies with synthetic tubulin peptides

The interaction of microtubule-associated proteins MAP-1 and MAP-2 with different peptides containing sequences covering the C-terminal region of beta-tubulin isoforms has been analyzed. Our results indicate that MAP-1 and MAP-2 bind to a common sequence within the variable C-terminal region of the different beta-tubulin isoforms, while MAP-2 also interacts with the subdomain beta (422-434) of the constant region, in agreement with previous results (Maccioni, R.B., Rivas, C., & Vera, J.C. (1988) EMBO J. 7, 1957-1963). The productive interaction of MAP-2 with the latter domain appears to be involved in the assembly of microtubules.

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val 187Gly 204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides α(430–441) and β(422–434). The binding data showed a relatively stronger interaction of Val 187-Gly 204 with β(422–434) as compared to that with α(430–441). The interaction of this tau peptide with either α or β tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The β tubulin peptide also appears to induce a structural change of tau fragment Val 218Gly 235. Interestingly, tau peptides Val 187Gly 204 and Val 218Gly 235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by antiidiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the α(430–441) and β(422–434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.

Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins.

In previous studies we have demonstrated that a 4-kd tubulin fragment, including amino acid residues from Phe418 to Glu450 in alpha-subunit and Phe408-Ala445 of the beta-sequence, plays a major role in controlling tubulin interactions leading to microtubule assembly. The 4-kd carboxyl-terminal domain also constitutes an essential domain for the interaction of microtubule-associated proteins (MAPs). Removal of the 4-kd fragment facilitates tubulin self-association and renders the assembly MAP-independent. In order to define the substructure of the tubulin domain for MAP interaction, we have examined the binding of 3H-acetylated C-terminal peptides to MAP-2 and tau. Two synthetic peptides from the low-homology region within the 4-kd domain alpha (430-441) and beta (422-434) and the peptide, alpha (401-410) of the high-homology region adjacent to the 4-kd domain, were analyzed with respect to MAP interaction. The binding data showed a relatively strong interaction of MAP-2 with the beta (422-434) peptide and a weaker interaction of both MAPs components with alpha (430-441) tubulin peptide as analyzed by Airfuge ultracentrifugation and zone filtration chromatography. The homologous alpha (401-410) peptide did not bind to either MAP-2 or tau. Equilibrium dialysis experiments showed a co-operative binding of beta (422-434) peptide to multiple sites in tau. The alpha (430-441) peptide exhibited a stronger interaction for tau as compared with MAP-2.(ABSTRACT TRUNCATED AT 250 WORDS)

A Proton Magnetic Resonance and a Circular Dichroism Study of the Solvent Dependent Conformation of the Synthetic Tubulin Fragment Ac Tubulin, Alpha (430–441) Amide and its Interaction with Substance-P

Proton magnetic resonance techniques were used to study the conformation of the synthetic tubulin fragment Ac-tubulin (430-441) amide in H2O and 80% CD3OH/20% D2O solutions, using water suppression techniques. Proton assignments are based on two-dimensional COSY experiments combined with one-dimensional spin decoupling. A comparison of the NH proton shifts between the two solvents, namely delta(CD3OH/H2O-H2O) shows a small solvent effect for the Lys1 to Val6 region of the molecule, whereas for Gly7 to Glu12 the solvent effect is much larger. The smaller effects in the region of Lys1 to Val6 may be due to some hydrogen bonding as these protons are shielded from the solvent. These conclusions are in agreement with the circular dichroism results in 80% methanol/20% water where the alpha helix is present to the extent of 30%, whereas the peptide is completely unstructured in water with some aggregation. The temperature dependence of the NH proton shifts was also carried out. In water these shifts are of the order of 7-9 X 10(-3) ppm/K indicating that most of the protons are not involved in hydrogen bonding. In CD3OH/H2O, these values range from about 4-6 X 10(-3) ppm/K, which are compatible with the presence of hydrogen bonds. Finally, binding studies were carried out between the tubulin peptide and the undecapeptide neutrotransmitter substance P. The largest shifts are for the Tyr3 NH proton of the tubulin fragment, whereas for substance P it is for the Lys3, Gln5 and Leu10 NH protons, indicating a change in conformation of both peptides on interaction.

ENZYMATIC ACTIVITY IN TUBULIN PREPARATIONS: CYCLIC‐AMP DEPENDENT PROTEIN KINASE ACTIVITY OF BRAIN MICROTUBULE PROTEIN1

Abstract—Cyclic‐AMP stimulates phosphorylation of Hf1 and Hf2b histone fractions and of protamine by tubulin preparations. Lyophilization of the tubulin removes the requirement for cyclic‐AMP; the kinase is fully active in the absence of cyclic nucleotide. The casein kinase activity of tubulin is independent of cyclic‐AMP. Only cyclic‐IMP can replace cyclic‐AMP at the low concentrations at which the cyclic‐AMP is effective. 5′‐AMP inhibits basal levels of Hf 1 histone phosphorylation. Several attempts have been made to separate the kinase activity from tubulin. Both tubulin and kinase activity are precipitated by vinblastine. Mg2+ precipitation of tubulin inactivates the kinase; there is no evidence that the precipitation step removes the kinase from tubulin. Tubulin which has been assembled into microtubules, collected by centrifugation and disassembled, retains its kinase activity. Kinase activity and tubulin co‐elute from DEAE‐cellulose. Taken together, these experiments demonstrate that tubulin, prepared by fractionation with ammonium sulfate followed by elution from DEAE Sephadex with 0‐8 M KC1 and concentration by reprecipitation with ammonium sulfate has a closely associated, cyclic‐AMP dependent protein kinase activity, which may be intrinsic to the tubulin peptides themselves.