Regulation of Op18 during spindle assembly in Xenopus egg extracts.
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.
Budde
P P
PP
Department of Molecular & Cell Biology, University of California, Berkeley, 94720, USA.
Kumagai
A
A
Dunphy
W G
WG
Heald
R
R
D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D008868:Microtubule Proteins; D010063:Ovum / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D008941:Spindle Apparatus / Q000502:physiology*; D050777:Stathmin; D014981:Xenopus; D029867:Xenopus Proteins; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D002843:Chromatin; D008868:Microtubule Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D050777:Stathmin; D029867:Xenopus Proteins; C493996:STMN1 protein, Xenopus; C101085:Plk1 protein, Xenopus; D000097869:Polo-Like Kinase 1