The stathmin phosphoprotein family: intracellular localization and effectson the microtubule network

Stathmin is a small regulatory phosphoprotein integrating diverse intracell ular signaling pathways, It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variant s RB3' and RB3", Stathmin itself was shown to interact in vitro with tubuli n in a phosphorylation-dependent manner, sequestering free tubulin and henc e promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known memb ers of the stathmin family, Whereas stathmin is not associated with interph ase microtubules in HeLa cells, a fraction of it is concentrated at the mit otic spindle, We generated antisera specific for stathmin phosphoforms, whi ch allowed us to visualize the regulation of phosphorylation-dephosphorylat ion during the successive stages of mitosis, and the partial localization o f stathmin phosphorylated on serine 16 at the mitotic spindle, Results from overexpression experiments of wildtype and novel phosphorylation site muta nts of stathmin further suggest that it induces depolymerization of interph ase and mitotic microtubules in its unphosphorylated state but is inactivat ed by phosphorylation in mitosis, Phosphorylation of mutants 16A25A and 38A 63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the f ormation of a functional spindle, whereas mutant 16A25A38A63E retained a mi crotubule depolymerizing activity, Transient expression of each of the neur al phosphoproteins of the stathmin family showed that they are at least par tially associated to the Golgi apparatus and not to other major membrane co mpartments, probably through their different NH2-terminal domains, as descr ibed for SCG10. Most importantly, like stathmin and SCG10, overexpressed SC LIP, RB3 and RB3" were able to depolymerize interphase microtubules, Altoge ther, our results demonstrate in vivo the functional conservation of the st athmin domain within each protein of the stathmin family, with a microtubul e destabilizing activity most likely essential for their specific biologica l function(s).