Interaction of Kinesin Motor Domains with α- and β-Tubulin Subunits at a Tau-independent Binding Site

Abstract

Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both alpha- and beta-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.

The Tubulin Code

“…75 In vitro, Tau, MAP1B, and MAP2 bind preferentially to tubulins with moderate levels of polyglutamylation (~3 glutamyl units) whereas MAP1A shows optimal affinity for highly modified tubulins (~6 glutamyl units). [76][77][78] As a-tubulin glutamylation is abundant in very young neurons whereas b-tubulin glutamylation increases during post-natal development, 50 glutamylation could control transitions in MAP binding during neuronal development. 78 Lys 40 a-tubulin acetylation may also influence MAP binding as overexpression of HDAC6 delocalized p58, a MAP involved in the association of Golgi membranes with microtubules.…”

“…91,92 Gel overlay and antibody inhibition experiments have shown that Kinesin-1 also binds preferentially to tubulin containing 3 glutamyl units. 77 To directly examine the influence of PTMs on motors, recent experiments have utilized microtubules lacking specific PTMs due to genetic ablation of either the PTM sites or enzymes. Reed et al showed that loss of a-tubulin acetylation, a-tubulin detyrosination, or b-tubulin polymodifications resulted in decreased binding of Kinesin-1 to microtubules whereas loss of a-tubulin polymodifications had no effect.…”

Muscle Creatine Kinase Deficiency Triggers Both Actin Depolymerization and Desmin Disorganization by Advanced Glycation End Products in Dilated Cardiomyopathy

“…Western Blot Analysis-One-dimensional and two-dimensional PAGE were carried out as described previously (15,16). Proteins were transferred onto nitrocellulose membranes (Hybond C, Amersham Biosciences) and the blots were saturated in TBS-T buffer (20 mM Tris, pH 7.5, 136.8 mM NaCl, and 0.1% (v/v) Tween 20) containing 5% (w/v) low fat milk.…”

Loss of α-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function

“…The binding affinity of kinesin motors to microtubules in vitro can be affected by the extent of tubulin polyglutamylation (5,24). To investigate whether ␣-tubulin polyglutamylation affects binding of kinesins to microtubules, we analyzed the ability of kinesins to copurify with microtubules isolated from brains of ROSA22 mutant and control mice (Fig.…”