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 NIH 3T3 fibroblasts expressing a Raf-1 kinase domain-estrogen receptor fusion protein (BXB-ER), whose activity can be acutely regulated by estrogen. Proteins differentially phosphorylated 20 min after BXB-ER activation in living cells were displayed by two-dimensional electrophoresis. The protein with the most prominent newly induced phosphorylation was identified as stathmin, a phosphorylation-sensitive regulator of microtubule dynamics. Stathmin is rapidly phosphorylated on two ERK phosphorylation sites (serines 25 and 38) upon BXB-ER activation. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase (MEK) inhibitor PD98059 abolished this phosphorylation, demonstrating that stathmin is targeted by BXB-ER via the MEK/ERK pathway. Prolonged BXB-ER activation resulted in the accumulation of a stathmin phosphoisomer with impaired microtubule-destabilizing activity. The appearance of this phosphoisomer after BXB-ER activation correlated with rearrangements in the microtubule network, resulting in the formation of long bundled microtubules extending toward the rim 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-mediated stathmin phosphorylation plays an important role for the microtubule reorganization induced by acute activation of Raf-1.
Institut für Klinische Molekularbiologie und Tumorgenetik der GSF, Marchioninistrasse 25, D-81377 Munich, Germany. Lovric@gsf.de
J
J
Lovrić
S
S
Dammeier
A
A
Kieser
H
H
Mischak
W
W
Kolch
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry; D051379:Mice; D008868:Microtubule Proteins; D008870:Microtubules / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D050777:Stathmin
D008868:Microtubule Proteins; D010750:Phosphoproteins; D050777:Stathmin; C493994:Stmn1 protein, mouse; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf