Regulation of Op18 during spindle assembly in Xenopus egg extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negati vely regulated by phosphorylation. To evaluate the role of the three Op18 p hosphorylation sites in Xenopus (Ser 16, 25, and 39). we added wild-type Op 18, 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 le ngth and density, while Op18-EEE did not significantly affect spindle micro tubules. Affinity chromatography with these proteins revealed that the micr otubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mi totic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary char acterization of the chromatin proteins recruited to DNA beads, and identifi ed the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induce d Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Pl x1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.