Regulation of Op18 during Spindle Assembly in Xenopus Egg Extracts
Abstract
Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.
Additional Information
© The Rockefeller University Press 2001 Submitted: 26 October 2000. Revised: 14 February 2001. Accepted: 14 February 2001 We are grateful to S. Andersen for Op18 constructs, I. Vernos, T. Hirano, and J. Ferrell for antibodies to Xklp1, Topoisomerase II, and MAP kinase, respectively. We thank T. Blank for help with micrococcal nuclease digests, J. Swedlow for advice regarding kinase assays, N. Rao and R. Yue for help generating Op18 constructs, A. Desai for a frog tubulin purification protocol, and G.O. Nads for sperm nuclei. We also thank members of the Heald, Welch, and Weis labs for helpful discussion, and Matthew Welch, Sarah Wignall, and Jennifer Banks for critical reading of the manuscript. R. Heald is supported by the National Institutes of Health, The Pew Charitable Trust, and the Cancer Research Coordinating Committee.Files
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2006-09-18Created from EPrint's datestamp field
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2021-11-08Created from EPrint's last_modified field