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. 2010 Feb 5;285(6):3548-53.
doi: 10.1074/jbc.M109.064113. Epub 2009 Dec 3.

Regulation of Neurite Outgrowth by Interactions Between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-Associated Protein Superior Cervical Ganglia Clone 10

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Free PMC article

Regulation of Neurite Outgrowth by Interactions Between the Scaffolding Protein, JNK-associated Leucine Zipper Protein, and Neuronal Growth-Associated Protein Superior Cervical Ganglia Clone 10

Hua Xu et al. J Biol Chem. .
Free PMC article

Abstract

JLP (JNK-associated leucine zipper protein) is a novel scaffolding protein involved in JNK signaling. Although it is known that JLP is highly expressed in brain, the biological function of JLP in neuronal systems remains unknown. Here, we report a novel interaction between JLP and SCG10 (superior cervical ganglia clone 10), which is a microtubule-destabilizing factor that is essential for neurite outgrowth. Inhibition of endogenous JLP expression using small interference RNA methodology strongly enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Our results show that JLP negatively regulates NGF-induced neurite outgrowth by decreasing the level of phosphorylated SCG10. Furthermore, inhibition of JNK phosphorylation by a small molecule inhibitor, SP600125, resulted in inhibition of SCG10 phosphorylation and inhibition of neurite growth. Taken together, our results suggest that JLP negatively regulates NGF-induced neurite outgrowth through a sequestering mechanism that results in an attenuation of NGF-induced SCG10 phosphorylation.

Figures

FIGURE 1.
FIGURE 1.
Interaction between JLP and stathmin family members. A, cell lysates derived from COS-7 cells overexpressing JLP-S and HA-tagged stathmin, SCG10, and SCLIP were subjected to pulldown assays using S-protein-agarose beads, and the precipitates were analyzed by SDS-PAGE, followed by Western blotting with an anti-HA antibody (middle panel). The expression of HA-tagged stathmin, SCG10, and SCLIP, as well as the precipitated S-tagged JLP (JLP-S), are shown in the top and bottom panels. B and C, schematic representation of the deletion and domain mutants of JLP, respectively. D and E, domain mapping of the JLP-SCG10 interaction. A series of deletion mutants (B) and domain mutants (C) of JLP were co-transfected into COS-7 cells with HA-SCG10 (top panel) and subjected to precipitation using S-protein-agarose. Precipitates were analyzed by SDS-PAGE, followed by Western blotting with an anti-HA antibody. The expression of HA-SCG10 and the precipitated JLP mutants are shown in the top and bottom panels.
FIGURE 2.
FIGURE 2.
SCG10 binds to JLP only in its full-length form. A, schematic representation of the domain mutants of SCG10. B, mapping JLP-interacting domain of SCG10. A series of domain mutants of SCG10 (top panel) were co-transfected into COS-7 cells with JLP-S and subjected to precipitation using S-protein-agarose. Precipitates were analyzed by SDS-PAGE, followed by Western blotting with an anti-HA antibody. The expression of HA-SCG10 mutants and the precipitated JLP are shown in the top and bottom panels. C, the interaction between JLP and SCG10 during NGF-induced differentiation of PC12 cells. An immunoprecipitation assay using an anti-SCG10 or JLP antibody was performed using cell lysates derived from the PC12 cells treated with NGF for 3 days. The immunoprecipitates together with the lysate were analyzed using the antibodies against JLP or SCG10. D, a time-course study of the interaction between JLP and SCG10. Immunoprecipitation assays were performed as in C using cell lysates derived from PC12 cells treated with NGF at various time points as indicated. The lysates were immunoprecipitated with control antibody (cont. Ab) or anti-SCG10 antibody (SCG10 Ab). Western blot analysis was performed on the lysates and the immunoprecipitates using specific antibodies against JLP and SCG10.
FIGURE 3.
FIGURE 3.
JLP regulates NGF-induced neurite outgrowth in PC12 cells. A, JLP protein levels in JLP siRNA-expressing cells (C, control siRNA; J, JLP siRNA). B, neurite outgrowth in NGF-treated PC12 cells in which endogenous JLP was down-regulated by its siRNA. The fields were randomly selected on the indicated day of NGF treatment. C, the percentage of differentiated PC12 cells undergoing NGF treatment. The differentiated cells were counted as described under “Experimental Procedures” (p < 0.001). D, neurite length of differentiated PC12 cells. The length of the longest neurite of each differentiated cell was measured using ImageJ software (National Institutes of Health).
FIGURE 4.
FIGURE 4.
The role of JLP in regulating SCG10 phosphorylation in PC12 cells. A, the phosphorylation level of endogenous SCG10 in PC12 cells in response to NGF treatment. PC12 cells were transfected with control siRNA (C) or JLP siRNA (J), followed by NGF treatment. Two days after the treatment, the cultures were incubated with [32P]orthophosphate for 2 h. Lysates from the radiolabeled cells were subjected to immunoprecipitation analysis using an anti-SCG10 antibody. The immunoprecipitates were resolved by SDS-PAGE and subjected to autoradiography. B, quantitation of SCG10 phosphorylation in the presence or absence of JLP. The top and bottom SCG10 protein bands indicated in A are quantitated separately. The basal phosphorylation levels of SCG10 in intact PC12 cells were defined as 1 unit.
FIGURE 5.
FIGURE 5.
JNK activity is required for the NGF-induced neurite outgrowth of PC12 cells. A, the effect of JNK inhibitor SP600125 on NGF-induced differentiation of PC12 cells. PC12 cells were incubated with vehicle (DMSO) or the JNK inhibitor (10 μm) 2 h before NGF treatment. Three days after the treatment, randomly selected fields were photographed in the indicated culture plates. B, changes in the phosphorylation level of SCG10 as a result of SP600125 treatment. PC12 cells were incubated with vehicle (DMSO) or the JNK inhibitor (10 μm) 2 h before NGF treatment. Three days after the treatment, the cultures were incubated with [32P]orthophosphate for 2 h. Lysates from the radiolabeled cells were immunoprecipitated using an anti-SCG10 antibody. The immunoprecipitates were resolved by SDS-PAGE and subjected to autoradiography. Phosphorylated levels of the top and bottom bands of SCG10 were quantitated. The phosphorylated levels of SCG10 of the control samples were set at 100. C, protein analysis of the lysates derived from the cells treated with vehicle (DMSO) or SP600125. PC12 cells were incubated with vehicle (DMSO) or the JNK inhibitor (10 μm) 2 h before NGF treatment. Three days after the treatment, the lysates were analyzed by Western blotting using specific antibodies against phospho-JNK, JNK, SCG10, JLP, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

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