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Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex.

Lalli G, Hall A - J. Cell Biol. (2005)

Bottom Line: Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching.In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons.Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, England, UK.

ABSTRACT
Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

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Ral regulates GAP-43 phosphorylation. (A) COS cells were transfected with the indicated myc-tagged Ral constructs and cGAP-43. Levels of phospho-cGAP-43 in cell extracts were determined by immunoblotting with an anti–phospho-GAP-43 antibody. A representative blot is shown. Inactive Ral causes a significant decrease of cGAP-43 phosphorylation (top band, arrow). (B) Quantitative analysis of phosphorylated cGAP-43 in COS cells transfected with cGAP-43 and the indicated Ral constructs. Shown here are data from four independent experiments (means ± SEM; *, P < 0.01). (C) Cortical neurons were nucleofected with the indicated constructs and lysed after overnight expression. Levels of total and phosphoendogenous GAP-43 were then assessed by immunoblotting. The fourth, fifth, and sixth lanes were derived from the same blot. (D) Quantitative analysis of endogenous GAP-43 phosphorylation in cortical neurons after nucleofection with the indicated Ral mutant isoforms. Data were derived from four independent experiments (means ± SEM; **, P < 0.005).
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fig5: Ral regulates GAP-43 phosphorylation. (A) COS cells were transfected with the indicated myc-tagged Ral constructs and cGAP-43. Levels of phospho-cGAP-43 in cell extracts were determined by immunoblotting with an anti–phospho-GAP-43 antibody. A representative blot is shown. Inactive Ral causes a significant decrease of cGAP-43 phosphorylation (top band, arrow). (B) Quantitative analysis of phosphorylated cGAP-43 in COS cells transfected with cGAP-43 and the indicated Ral constructs. Shown here are data from four independent experiments (means ± SEM; *, P < 0.01). (C) Cortical neurons were nucleofected with the indicated constructs and lysed after overnight expression. Levels of total and phosphoendogenous GAP-43 were then assessed by immunoblotting. The fourth, fifth, and sixth lanes were derived from the same blot. (D) Quantitative analysis of endogenous GAP-43 phosphorylation in cortical neurons after nucleofection with the indicated Ral mutant isoforms. Data were derived from four independent experiments (means ± SEM; **, P < 0.005).

Mentions: In our search for molecules that could act downstream of Ral to promote branching, we focused on GAP-43, a neuronal protein known to promote the formation and growth of local branches in target regions of innervation (Aigner et al., 1995) but also able to induce filopodia in various cell types (Widmer and Caroni, 1993). Phosphorylation of GAP-43 on Ser41 by PKC and its membrane anchoring through palmitoylation of cysteine residues at the NH2 terminus promote cytoskeletal remodeling (Widmer and Caroni, 1993; Aigner et al., 1995). Because PKC-dependent phosphorylation at Ser41 is associated with sprout-promoting activity (Aigner et al., 1995) and adhesion to laminin stimulates GAP-43 phosphorylation (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200507061/DC1), we determined whether Ral could affect the phosphorylation state of this residue in GAP-43. COS cells were first cotransfected with Ral mutants and an epitope-tagged chick version of GAP-43 (cGAP-43; Widmer and Caroni, 1993). In chick, the PKC phosphorylation site of GAP-43 is Ser42, corresponding to Ser41 in mammalian GAP-43 (Widmer and Caroni, 1993). Levels of phosphorylated cGAP-43 were then monitored by immunoblotting (Fig. 5 A). Dominant-negative Ral substantially decreased GAP-43 phosphorylation. In contrast, active Ral had little effect (Fig. 5 B), possibly because of intrinsically high levels of PKC activity in COS cells. To test the ability of Ral to modulate GAP-43 phosphorylation in primary neurons, we measured levels of endogenous phosphorylated GAP-43 in cortical neurons 24 h after nucleofection with Ral mutants. Efficiency of nucleofection in these cells was between 50 and 70%, and myc-tagged Ral was detected by immunoblotting (Fig. 5 C). Quantitative analysis showed that dominant-negative Ral significantly decreased phosphorylation of endogenous GAP-43, whereas active Ral caused a threefold increase (Fig. 5 D). In addition, phosphorylation of endogenous GAP-43 significantly decreased in neurons depleted of Ral by nucleofection of RalA and -B siRNA (Fig. S4 A). Finally, treatment with the PKC inhibitor GF109203X completely blocked the enhanced phosphorylation of endogenous GAP-43 caused by active Ral (Fig. S4 B). We conclude that Ral regulates PKC-mediated phosphorylation of GAP-43 on Ser41.


Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex.

Lalli G, Hall A - J. Cell Biol. (2005)

Ral regulates GAP-43 phosphorylation. (A) COS cells were transfected with the indicated myc-tagged Ral constructs and cGAP-43. Levels of phospho-cGAP-43 in cell extracts were determined by immunoblotting with an anti–phospho-GAP-43 antibody. A representative blot is shown. Inactive Ral causes a significant decrease of cGAP-43 phosphorylation (top band, arrow). (B) Quantitative analysis of phosphorylated cGAP-43 in COS cells transfected with cGAP-43 and the indicated Ral constructs. Shown here are data from four independent experiments (means ± SEM; *, P < 0.01). (C) Cortical neurons were nucleofected with the indicated constructs and lysed after overnight expression. Levels of total and phosphoendogenous GAP-43 were then assessed by immunoblotting. The fourth, fifth, and sixth lanes were derived from the same blot. (D) Quantitative analysis of endogenous GAP-43 phosphorylation in cortical neurons after nucleofection with the indicated Ral mutant isoforms. Data were derived from four independent experiments (means ± SEM; **, P < 0.005).
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fig5: Ral regulates GAP-43 phosphorylation. (A) COS cells were transfected with the indicated myc-tagged Ral constructs and cGAP-43. Levels of phospho-cGAP-43 in cell extracts were determined by immunoblotting with an anti–phospho-GAP-43 antibody. A representative blot is shown. Inactive Ral causes a significant decrease of cGAP-43 phosphorylation (top band, arrow). (B) Quantitative analysis of phosphorylated cGAP-43 in COS cells transfected with cGAP-43 and the indicated Ral constructs. Shown here are data from four independent experiments (means ± SEM; *, P < 0.01). (C) Cortical neurons were nucleofected with the indicated constructs and lysed after overnight expression. Levels of total and phosphoendogenous GAP-43 were then assessed by immunoblotting. The fourth, fifth, and sixth lanes were derived from the same blot. (D) Quantitative analysis of endogenous GAP-43 phosphorylation in cortical neurons after nucleofection with the indicated Ral mutant isoforms. Data were derived from four independent experiments (means ± SEM; **, P < 0.005).
Mentions: In our search for molecules that could act downstream of Ral to promote branching, we focused on GAP-43, a neuronal protein known to promote the formation and growth of local branches in target regions of innervation (Aigner et al., 1995) but also able to induce filopodia in various cell types (Widmer and Caroni, 1993). Phosphorylation of GAP-43 on Ser41 by PKC and its membrane anchoring through palmitoylation of cysteine residues at the NH2 terminus promote cytoskeletal remodeling (Widmer and Caroni, 1993; Aigner et al., 1995). Because PKC-dependent phosphorylation at Ser41 is associated with sprout-promoting activity (Aigner et al., 1995) and adhesion to laminin stimulates GAP-43 phosphorylation (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200507061/DC1), we determined whether Ral could affect the phosphorylation state of this residue in GAP-43. COS cells were first cotransfected with Ral mutants and an epitope-tagged chick version of GAP-43 (cGAP-43; Widmer and Caroni, 1993). In chick, the PKC phosphorylation site of GAP-43 is Ser42, corresponding to Ser41 in mammalian GAP-43 (Widmer and Caroni, 1993). Levels of phosphorylated cGAP-43 were then monitored by immunoblotting (Fig. 5 A). Dominant-negative Ral substantially decreased GAP-43 phosphorylation. In contrast, active Ral had little effect (Fig. 5 B), possibly because of intrinsically high levels of PKC activity in COS cells. To test the ability of Ral to modulate GAP-43 phosphorylation in primary neurons, we measured levels of endogenous phosphorylated GAP-43 in cortical neurons 24 h after nucleofection with Ral mutants. Efficiency of nucleofection in these cells was between 50 and 70%, and myc-tagged Ral was detected by immunoblotting (Fig. 5 C). Quantitative analysis showed that dominant-negative Ral significantly decreased phosphorylation of endogenous GAP-43, whereas active Ral caused a threefold increase (Fig. 5 D). In addition, phosphorylation of endogenous GAP-43 significantly decreased in neurons depleted of Ral by nucleofection of RalA and -B siRNA (Fig. S4 A). Finally, treatment with the PKC inhibitor GF109203X completely blocked the enhanced phosphorylation of endogenous GAP-43 caused by active Ral (Fig. S4 B). We conclude that Ral regulates PKC-mediated phosphorylation of GAP-43 on Ser41.

Bottom Line: Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching.In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons.Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, England, UK.

ABSTRACT
Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C-dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

Show MeSH
Related in: MedlinePlus