Interaction of tubulin and microtubule proteins with vanadate oligomers.
Microtubule assembly is known to be regulated by the phosphorylation of microtubule-associated proteins (MAPs), and is thus sensitive to phosphatase inhibitors. We have investigated the direct interaction between phosphatase inhibitors (vanadate, sodium fluoride, and okadaic acid) and microtubule proteins. Vanadate self-assembles into oligomers, primarily dimer, tetramer, and decamer in 0.1 M Pipes, pH 6.9. Oligomer concentrations and their direct binding to tubulin and MAPs were determined by 51V NMR. The assembly of microtubule protein (MTP) is strongly inhibited by decavanadate binding to MAPs and only weakly inhibited by tetravanadate binding to MAPs. Decavanadate will inhibit both MAP2 and tau-induced assembly. Decavanadate binds to MAP2 at 26 sites [Ka > or = (1.0-1.3) x 10(5) M-1]. The mechanism appears to involve competitive binding to MAPs, presumably at or near the microtubule binding domains, and reduced affinity for microtubules. The assembly of MAP-free, phosphocellulose-purified tubulin (PC-tubulin) is only weakly inhibited by decavanadate, although decavanadate binds to tubulin at four independent sites (Ka > or = 1.0 x 10(5) M-1). Monomeric vanadate, a strong phosphatase inhibitor, does not interact with tubulin or MAPs, and thus does not bind to the exchangeable nucleotide binding site on tubulin. Sodium fluoride stimulates both PC-tubulin and MTP assembly by a nonspecific effect, probably involving water structure formation. Wyman analysis suggests an absence of direct or specific binding to tubulin (d ln K/d ln [NaF] = 0.214). NaCl is nearly as effective in promoting assembly of PC-tubulin, but inhibits MTP assembly.(ABSTRACT TRUNCATED AT 250 WORDS)
Lobert
S
S
School of Nursing, University of Mississippi Medical Center, Jackson 39216.
Isern
N
N
Hennington
B S
BS
Correia
J J
JJ
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001923:Brain Chemistry; D002482:Cellulose / Q000031:analogs & derivatives; D004988:Ethers, Cyclic / Q000494:pharmacology; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008868:Microtubule Proteins / Q000378:metabolism*; D019319:Okadaic Acid; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D012969:Sodium Fluoride / Q000494:pharmacology; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D004988:Ethers, Cyclic; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D014404:Tubulin; D019319:Okadaic Acid; D014638:Vanadates; D012969:Sodium Fluoride; D002482:Cellulose; C008881:phosphocellulose; D010744:Phosphoric Monoester Hydrolases