Abstract
Nucleophosmin/B23, an abundant nucleolar protein, plays multiple roles in cell growth and proliferation, and yet, little has been studied about its function in regulating dynamics of microtubules. Here, we report that B23 directly interacts with Eg5, a member of the kinesin family, in the cytosol. The DNA/RNA binding domain of B23 and the motor domain of Eg5 were found to be involved in their interaction. Both in vivo and in vitro evidences showed that B23 acts as an upstream regulator of Eg5 in promoting microtubule polymerization. Moreover, we further demonstrated that B23 regulates microtubule dynamics by directly inhibiting Eg5 ATPase activity.
Highlights
19060 JOURNAL OF BIOLOGICAL CHEMISTRY provokes cell proliferation and transformation, and B23 is frequently mutated in many different tumor types such as acute myeloid leukemia (AML) (9 –11)
We identified that B23/nucleophosmin interacts directly with Eg5, and through this interaction, B23 inhibited ATPase activity of the Eg5 motor domain and down-regulated the Eg5-mediated ATP-dependent microtubule catastrophe
Stable and dynamic MTs were fractionated for each treated cell, and the results showed that knockdown of Eg5 decreases dynamic MTs (Fig. 4a, lanes 1 and 2 versus lanes 3 and 4); knockdown of B23, in contrast, increases dynamic MTs (Fig. 4a, lanes 1 and 2 versus lanes 5 and 6)
Summary
Plasmid Clones and Protein Purification—Total RNA was purified from HeLa cells using TRIzol reagent (Invitrogen). His6-Eg5-(1–111) was constructed by inserting its PCR fragment into the EcoRI/XhoI sites of the pET22b(ϩ) plasmid These proteins were purified as described previously [15]. Stained cells were mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA) and analyzed under an Olympus IX71 inverted microscope coupled to an Olympus DP70 high resolution color camera. Yeast Two-hybrid Assay—The B23 open reading frame was cloned into the pGBK-T7 plasmid and was used as bait in a yeast two-hybrid screen of a HeLa cDNA library (Clontech) as described previously [15]. Cells were washed twice by PEM buffer and incubated with PEM supplemented with 0.05% Triton X-100 at 37 °C for 30 min. Fluorescence and differential interference contrast images were obtained every 5 min for a period of 2–3 h
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