Figure 2.

Nek2 binds microtubules in vitro. (A) Nek2A and control proteins, Rab4 and γ-tubulin, were generated by in vitro translation in the presence of [³⁵S]methionine. These proteins (Input) were incubated without (–MT) or with (+MT) purified microtubules in the presence of Taxol and AMP-PNP, unless indicated. Proteins were then spun at 35,000 rpm on a sucrose cushion for 30 min. Pellet (P) and supernatant (S) fractions were collected and analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography. The Coomassie Blue–stained gel with Nek2A samples is shown, with tubulin being the predominant band. (B) Schematic representation of Nek2 constructs, indicating amino acid positions (CAT, catalytic domain; LZ, leucine zipper motif; CC, coiled-coil). The C-terminus of Nek2B (black) differs from Nek2A because of a splice site after residue 370. (C) These constructs were used in independent microtubule pull-down assays as detailed above. The presence of pelleted microtubules is shown by Coomassie Blue staining of the gels (right panels) before autoradiography (left panels). The histogram indicates the percentage of input protein found within the pellet fractions with (gray) and without (black) microtubules. (D) Amino acid sequence of the putative microtubule binding motif from human (Hs), mouse (Mm), and Xenopus laevis (Xl) Nek2 with amino acid positions indicated; complete match (*), conservative substitution in one sequence (:), divergent substitution in one sequence (.).