Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.

SCG10 is a neuronal growth-associated protein that is concentrated in the growth cones of developing neurons. SCG10 shows a high degree of sequence homology to the ubiquitous phosphoprotein stathmin, which has been recently identified as a factor that destabilizes microtubules by increasing their catastrophe rate. Whereas stathmin is a soluble cytosolic protein, SCG10 is membrane-associated, indicating that the protein acts in a distinct subcellular compartment. Identifying the precise intracellular distribution of SCG10 as well as the mechanisms responsible for its specific targeting will contribute to elucidating its function. The main structural feature distinguishing the two proteins is that SCG10 contains an NH 2 -terminal extension of 34 amino acids. In this study, we have examined the intracellular distribution of SCG10 in PC12 cells and in transfected COS-7 cells and the role of the NH 2 -terminal domain in membrane-binding and intracellular targeting. SCG10 was found to be localized to the Golgi complex region. We show that the NH 2 -terminal region (residues 1-34) was necessary for membrane targeting and Golgi localization. Fusion proteins consisting of the NH 2 -terminal 34 amino acids of SCG10 and the related protein stathmin or the unrelated protein, ␤-galactosidase, accumulated in the Golgi, demonstrating that this sequence was sufficient for Golgi localization. Biosynthetic labeling of transfected COS-7 cells with [ 3 H]palmitic acid revealed that two cysteine residues contained within the NH 2 -terminal domain were sites of palmitoylation.

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles. In isolated growth cone particles, SCG10 was found to be particularly concentrated in the growth cone vesicle fraction. To evaluate the molecular determinants of the specific targeting of SCG10 to growth cones, we have transfected PC12 cells and primary neurons in culture with mutant and fusion cDNA constructs. Deletion of the amino-terminal domain or mutations within this domain that prevented palmitoylation at cysteines 22 and 24 abolished Golgi localization as well as growth cone targeting, suggesting that palmitoylation of the amino-terminal domain is a necessary signal for Golgi sorting and possibly transport of SCG10 to growth cones. Fusion proteins consisting of the amino-terminal domain of SCG10 and the cytosolic proteins stathmin or glutathione-S-transferase colocalized with a Golgi marker, alpha-mannosidase II, and accumulated in growth cones of both axons and dendrites. These results reveal a novel axonal/dendritic growth cone targeting sequence that involves palmitoylation.

Abstract. Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.