The catastrophe-promoting activity of ectopic Op18/stathmin is required for disruption of mitotic spindles but not interphase microtubules.

Abstract

Oncoprotein18/stathmin (Op18) is a microtubule (MT) destabilizing protein that is inactivated during mitosis by phosphorylation at four Ser-residues. Op18 has at least two functions; the N-terminal region is required for catastrophe-promotion (i.e., transition from elongation to shortening), while the C-terminal region is required to inhibit MT-polymerization rate in vitro. We show here that a "pseudophosphorylation" derivative of Op18 (i.e., four Ser- to Glu-substitutions at phosphorylation sites) exhibits a selective loss of catastrophe-promoting activity. This is contrasted to authentic phosphorylation, which efficiently attenuates all activities except tubulin binding. In intact cells, overexpression of pseudophosphorylated Op18, which is not phosphorylated by endogenous kinases, is shown to destabilize interphase MTs but to leave spindle formation untouched. To test if the mitotic spindle is sensitive only to the catastrophe-promoting activity of Op18 and resistant to C-terminally associated activities, N- and C-terminal truncations with defined activity-profiles were employed. The cell-cycle phenotypes of nonphosphorylatable mutants (i.e., four Ser- to Ala-substitutions) of these truncation derivatives demonstrated that catastrophe promotion is required for interference with the mitotic spindle, while the C-terminally associated activities are sufficient to destabilize interphase MTs. These results demonstrate that specific Op18 derivatives with defined activity-profiles can be used as probes to distinguish interphase and mitotic MTs.