ACTIVATED RAF INDUCES THE HYPERPHOSPHORYLATION OF STATHMIN AND THE REORGANIZATION OF THE MICROTUBULE NETWORK

Raf kinases are regulators of cellular proliferation, transformation, differentiation, and apoptosis. To identify downstream targets of Raf- 1 in vivo, we used NIR 3T3 fibroblasts expressing a Raf-1 kinase domai n-estrogen receptor fusion protein (BXB-ER), whose activity can be acu tely regulated by estrogen, Proteins differentially phosphorylated 20 min after BXB-ER activation in Living cells were displayed by two-dime nsional electrophoresis. The protein with the most prominent newly ind uced phosphorylation was identified as stathmin, a phosphorylation-sen sitive regulator of microtubule dynamics. Stathmin is rapidly phosphor ylated on two ERK phosphorylation sites (serines 25 and 38) upon BXB-E R activation. The mitogen-activated protein kinase/extracellular signa l-regulated kinase-kinase (MEK) inhibitor PD98059 abolished this phosp horylation, demonstrating that stathmin is targeted by BXB-ER via the MEK/ERK pathway. Prolonged BXB-ER activation resulted in the accumulat ion of a stathmin phosphoisomer with impaired microtubule-destabilizin g activity. The appearance of this phosphoisomer after BXB-ER activati on correlated with rearrangements in the microtubule network, resultin g in the formation of long bundled microtubules extending toward the r im of the cells. Our results identify stathmin as a main target of the Raf/MEK/ERK kinase cascade in vivo and strongly suggest that ERK-medi ated stathmin phosphorylation plays an important role for the microtub ule reorganization induced by acute activation of Raf-1.