PDZ-RhoGEF ubiquitination by Cullin3-KLHL20 controls neurotrophin-induced neurite outgrowth

Abstract

The induction of neurite outgrowth and arborization is critical for developmental and regenerative processes. In this paper, we report that the BTB-kelch protein KLHL20 promoted neurite outgrowth and arborization in hippocampal and cortical neurons through its interaction with Cullin3 to form a ubiquitin ligase complex. This complex targeted PDZ-Rho guanine nucleotide exchange factor (RhoGEF), a protein abundantly expressed in the brain, for ubiquitin-dependent proteolysis, thereby restricting RhoA activity and facilitating growth cone spreading and neurite outgrowth. Importantly, targeting PDZ-RhoGEF to KLHL20 required PDZ-RhoGEF phosphorylation by p38 mitogen-activated protein kinase. In response to p38-activating neurotrophins, such as brain-derived neurotrophic factor and neurotrophin-3, KLHL20-mediated PDZ-RhoGEF destruction was potentiated, leading to neurotrophin-induced neurite outgrowth. Our study identified a ubiquitin-dependent pathway that targets PDZ-RhoGEF destruction to facilitate neurite outgrowth and indicates a key role of this pathway in neurotrophin-induced neuronal morphogenesis.

Figure 1.

KLHL20 promotes neurite outgrowth/arborization. (A) Confocal image of GFP-transfected DIV4 neurons stained with the anti-KLHL20 antibody (left). The relative intensities of the KLHL20 signal in axon and dendrites were quantified (see Materials and methods) and normalized by the intensity of GFP (right). Data represent means ± SEM from three independent experiments (***, P < 0.0005; n = 15). (B and C) Hippocampal neurons at DIV0 were transfected with the indicated plasmids together with GFP at a ratio of 4:1, and the morphologies of GFP-positive neurons were examined at DIV2 (B) or DIV5 (C). Representative morphologies of neurons are shown. Neurite length and various parameters of neuronal morphology (see Materials and methods) were quantified and plotted. (D) Cortical neurons were transfected as in B and monitored for neurite outgrowth at DIV4. Data in B–D represent means ± SEM from three independent experiments (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n ≥ 35). Bars, 50 µm.

Figure 2.

KLHL20 interacts with PDZ-RhoGEF. (A) Yeast cotransformed with the indicated constructs was assayed for the His3 phenotype (−His) and β-galactosidase activity (β-gal). (B and D) Interaction of PDZ-RhoGEF–Flag with KLHL20-Myc and mapping of their interaction domains. 293T cells transfected with various constructs were analyzed by immunoprecipitation (IP) and/or Western blotting (WB). The various deletion mutants of PDZ-RhoGEF are shown on the top of C. (C) GST pull-down analysis of the KLHL20 interaction with baculovirally purified PDZ-RhoGEF and its deletion mutants. The equal input of the GST fusion protein and the expression levels of various PDZ-RhoGEF mutants are shown on the bottom and right, respectively. (E) Coimmunoprecipitation analysis of the interaction between endogenous PDZ-RhoGEF and endogenous KLHL20 in the mouse brain. The dotted line indicates that unrelated lanes were removed. (F) KLHL20 mediates the interaction between Cul3 and PDZ-RhoGEF. HeLa cells infected with a lentivirus carrying KLHL20 siRNA or control siRNA were assayed by immunoprecipitation and/or Western blotting. (G) Coimmunoprecipitation analysis of the interaction of Cul3 with KLHL20 and PDZ-RhoGEF in cortical neurons.

Figure 3.

KLHL20 promotes PDZ-RhoGEF ubiquitination to inactivate RhoA. (A and B) KLHL20 promotes PDZ-RhoGEF ubiquitination in vivo. 293T cells (A) or HeLa cells stably expressing control or KLHL20 siRNA (B) were transfected with the indicated constructs and treated with MG132. Cells were lysed for immunoprecipitation (IP) and/or Western blot (WB) with the indicated antibodies. (C) PDZ-RhoGEF purified from baculovirus was subject to in vitro ubiquitination reaction in the presence of the E1, E2, and E3 complex and/or ubiquitin and then analyzed by Western blotting with the anti-Flag antibody. (D) Western blot analysis of the ectopic (left) or endogenous (right) level of PDZ-RhoGEF in 293T cells transfected with the indicated constructs and treated with or without MG132. (E) KLHL20 promotes PDZ-RhoGEF turnover. Western blot analysis of endogenous PDZ-RhoGEF level in 293T cells transfected with indicated constructs and treated with cycloheximide (CHX) for various time points. (F) Western blot analysis of endogenous PDZ-RhoGEF level in HeLa cells stably expressing indicated siRNA. (G–I) 293T cells (G), HeLa cells (H), or hippocampal neurons (I) were transfected with various constructs and/or siRNAs and then were lysed for assaying GTP-bound RhoA by pull-down (G and H) or G-LISA (I) or the expression of various proteins. Data in I represent means ± SEM (**, P < 0.005; n = 3). The efficacy of PDZ-RhoGEF siRNA to down-regulate the endogenous protein in HeLa cells is shown in Fig. S2 E. In D–F, the relative amounts of PDZ-RhoGEF were quantified, normalized to the amounts of tubulin, and marked below the blot. si, siRNA; Ub, ubiquitin.

Figure 4.

KLHL20 promotes neurite outgrowth and growth cone spreading through down-regulating PDZ-RhoGEF. (A and B) Hippocampal neurons at DIV0 were transfected with the indicated plasmids and/or siRNAs together with GFP. Neurons were imaged at DIV5 (A) or at DIV3, 4, and 5 (B), and neurite lengths of GFP-positive neurons were quantified and plotted. The efficacy of PDZ-RhoGEF siRNA to down-regulate endogenous PDZ-RhoGEF in hippocampal neurons is shown in Fig. S2 E. (C and D) Hippocampal neurons transfected with the indicated constructs and/or siRNAs were fixed at DIV3 and then stained by rhodamine-conjugated phalloidin and Tau-1 (not depicted). The percentage of axons showing collapsed growth cones was quantified and plotted (C, left). Representative growth cone morphologies are shown on the right. Data in all panels represent means ± SEM (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; n ≥ 50). Bars, 10 µm. WT, wild type.

Figure 5.

KLHL20-mediated PDZ-RhoGEF ubiquitination is stimulated by p38 MAPK and contributes in part to neurotrophin-induced morphogenesis. (A) GST pull-down analysis of the KLHL20 interaction with p38-phosphorylated or calf intestine phosphatase (CIP)–treated PDZ-RhoGEF. (B) Baculovirally purified PDZ-RhoGEF was phosphorylated by p38 and then subject to in vitro ubiquitination assay as in Fig. 3 C. (C) In vitro phosphorylation of PDZ-RhoGEF by p38. Substrate phosphorylation was analyzed by autoradiograph. (D and E) p38 inhibitor elevates PDZ-RhoGEF level (D) and RhoA activity (E) in hippocampal neurons. Hippocampal neurons at DIV1 were treated with 20 µM SB203580 for 3 h and then analyzed by Western blotting (WB) for protein expression (D) or G-LISA for RhoA activity (E, left). The expression of RhoA was determined by Western blotting (E, right). Data represent means ± SEM (**, P < 0.005; n = 3). (F and G) NT-3 (F) and BDNF (G) down-regulate PDZ-RhoGEF through p38 and KLHL20. Hippocampal neurons were transfected with control or KLHL20 siRNA at DIV0, treated at DIV2 with SB203580 and/or 200 ng/ml NT-3 or BDNF, and analyzed by Western blotting. (H and I) p38- and KLHL20-mediated PDZ-RhoGEF destruction participates in BDNF- and NT-3–induced differentiation. Hippocampal neurons were transfected with the indicated siRNAs and treated as in F. Neurite lengths were assayed at DIV4. Data represent means ± SEM (**, P < 0.005; ***, P < 0.0005; n ≥ 35). si, siRNA; Ub, ubiquitin.