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Published online 2 April 2001. doi:10.1083/jcb.153.1.149
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© The Rockefeller University Press, 0021-9525/2001//149 $5.00
The Journal of Cell Biology, Volume 153, Number 1, , 2001 149-158

Original Article

Regulation of Op18 during Spindle Assembly in Xenopus Egg Extracts



Priya Prakash Buddea, Akiko Kumagaib, William G. Dunphyb, and Rebecca Healda

a Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, California 94720
b Division of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125
311 LSA, Mail Code 3200, University of California, Berkeley, CA 94720.(510) 643-6791(510) 643-5493

heald{at}socrates.berkeley.edu

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.

Key Words: microtubule dynamics • spindle assembly • phosphorylation • Plx1 • chromatin

© 2001 The Rockefeller University Press

Abbreviations used in this paper: CSF, cytostatic factor-arrested; MAP, mitogen-activating protein; PP2A, protein phosphatase 2A; WT, wild type.


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