Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics t hat binds tubulin heterodimers and destabilizes MTs by promoting catastroph es (i.e., transitions from growing to shrinking MTs), Here, we have perform ed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in v itro, and (c) tubulin association and MT-regulating activities in intact ce lls, The data reveal distinct types of region-specific Op18 modulation of t ubulin GTP metabolism, namely inhibition of nucleotide exchange and stimula tion or inhibition of GTP hydrolysis. These regulatory activities are media ted via two-site cooperative binding to tubulin by multiple nonessential ph ysically separated regions of Op18. In vitro analysis revealed that NH2- an d COOH-terminal truncations of Op18 have opposite effects on the rates of t ubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both ca ses appeared independent of a simple tubulin sequestering mechanism. Howeve r, the NH2- and COOH-terminal-truncated Op18 mutants regulate MTs by distin ct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to reg ulate tubulin/MTs by more than one specific mechanism.