Isoform-specific palmitoylation of JNK regulates axonal development

Abstract

The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1-3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching.

Figure 1

JNK3 is palmitoylated at the COOH-terminus. (a) Metabolic labelling shows that JNK3 is palmitoylated in cortical neurons. JNK3 palmitoylation was examined by metabolic labelling with ³H-palmitic acid (³H-palm) (see Materials and Methods). Pre-treating neurons with 2-BrPA or adding HAM to JNK3 immunoprecipitates eliminated metabolic labelling signals. Normal rabbit IgG served as the immunoprecipitation control. The arrowhead indicates JNK3. (b) Palmitate on JNK3 has a short half-life. Pulse-chase experiments (chased at 0, 4, 6 h) show a short half-life (calculated ∼4.3 h, n=5) of palmitate on endogenous JNK3 in cortical neurons. (c) PATs show selectivity in palmitoylating JNK3 in heterogeneous HEK293 cells. PAT zD15 and zD20, but not zD23, enhances JNK3 palmitoylation, assessed by Btn-BMCC labelling (see Materials and Methods). (d) Two Cys residues at the COOH-terminus of JNK3 are critical for palmitoylation. The sequence alignment of the last 15 amino acids at the COOH-termini of JNK p54 isoforms reveals potential Cys residues (bold letters) for palmitoylation. Point-mutation of one or both Cys residues to Ser eliminates JNK3 palmitoylation, in the presence of PAT zD15. PAT activity-deficient zD15 (zD15Δ) loses the ability to promote JNK3 palmitoylation. (e) Palmitoylation occurs predominantly on the JNK3 isoform. The palmitoylation status of JNK p54 isoforms JNK1α2, JNK2α2 and JNK3α2, overexpressed in HEK293 cells, assessed by Btn-BMCC labelling, are shown and compared with the background (HAM minus). The schematic diagram at the bottom shows that JNK3 is the major isoform that is palmitoylated

Figure 2

The palmitoylation-deficient JNK3 mutant promotes axonal branching and filopodia motility. (a) Long and branched axons are observed in cultured hippocampal neurons at 7 DIV. Axons are labelled with the axonal marker Tau-1 (green) and DsRed2. Dendrites that are only labelled with DsRed2 are short and located near the somata. Scale bar: 10 μm. (b) Overexpression of the palmitoylation-deficient JNK3 mutant (JNK3 CS) increases axon length and branch complexity. Representative neurons transfected with DsRed2 alone, or together with JNK3 wild type (WT) or JNK3 CS, are shown. Scale bar: 10 μm. (c) Axonal branch numbers and total length of axons are enhanced by JNK3 CS. The tip numbers of secondary, tertiary and higher order of branches are shown. JNK3 CS increases the total branch number of axons (DsRed2 alone/blue, 5.0±0.2, n=131; WT/red, 5.6±0.3, n=66; CS/green, 10.6±0.4, n=81) and total axonal length (fold change to DsRed2 alone, WT, 1.1±0.1; CS, 1.4±0.1). Quantifications show mean±S.E.M. t-test. ^**P<0.01. (d) The density of axonal filopodia is not affected by JNK3 CS. Representative figures show axonal filopodia from transfected neurons. Scale bar: 5 μm. No significant changes in the CS group are observed (fold change to DsRed2 alone, WT, 1.0±0.1; CS, 1.0±0.1, P=0.85 to DsRed2 alone, P=0.40 to WT). (e) Axonal filopodia motility is promoted by JNK3 CS. Representative figures show the motility of axonal filopodia in WT and CS groups. The motility of axonal filopodia is further transformed into a single image and is shown below. Scale bar: 5 μm. Regions of marked dotted-squares are magnified and time-lapse images are shown with indicated time points within 135 s. Four labelled filopodia, as selected examples in the CS group, are more mobile than those in the WT group. Scale bar: 5 μm. The motility of axonal filopodia (10 filopodia per neuron from 20 neurons) is quantified with the MI (DsRed alone, 0.4±0.0, n=200; WT, 0.4±0.0, n=200, P=0.12; CS, 0.7±0.0, n=200, P<0.01)

Figure 3

Palmitoylation regulates the translocation of JNK3 to the Triton-insoluble actin cytoskeleton. (a) Palmitoylation does not affect JNK3 trafficking to lipid rafts. Lysates of HEK293 cells expressing the indicated constructs are subjected to density fractionation (see Materials and Methods). Fractions from Optiprep density-gradient centrfugation (f 1–13) are shown. Quantifications of the immunoblot intensity of the lipid raft marker caveolin-1 or JNKs from each fraction are shown at the bottom. Fractions 8 and 9 (f 8, 9) are marked as lipid rafts. Note the increase of JNK3 CS in fraction 13. (b) Endogenous neuronal JNK3 is present in the Triton X-100 insoluble-cytoskeleton fraction. In cortical neurons, incubation with Cyt.D or Lat.A reduces the presence of JNK3 in the insoluble fraction as calculated by the insoluble/soluble ratio (fold change to control, Cyt.D, 0.5±0.1; Lat.A, 0.4±0.11). Quantifications show mean±S.E.M. t-test. ^*P<0.05. Representative figures are from three independent experiments. (c), Palmitoylation regulates JNK3 translocation to the Triton X-100 insoluble fraction. The Triton X-100 solubility of JNK3 expressed in HEK293 was analyzed. The insoluble/soluble ratios of GFP-tagged JNK3 WT, JNK3 CS and JNK3 WT together with PAT zD15 are shown (WT, 0.7±0.1; CS, 1.5±0.2; WT+zD15, 0.3±0.1). t-test. ^**P<0.01

Figure 4

JNK3 palmitoylation modulates Wnt7a-regulated axonal branching. (a) JNK3 palmitoylation in neurons is regulated by Wnt7a. Brief application of Wnt7a (200 ng/ml, 6 h) to cortical neurons reduces JNK3 palmitoylation, assessed by Btn-BMCC labelling (fold change to control, Wnt7a, 0.6±0.1). Quantifications show mean±S.E.M. t-test. ^**P<0.01. The global palmitoylation status in cortical neurons is not changed. (b) Translocation of JNK3 to the Triton-insoluble fraction is regulated by Wnt7a. Wnt7a increases the presence of JNK3 in the insoluble fraction in cortical neurons (fold change to control, Wnt7a, 1.5±0.1). (c) Wnt7a-induced axon branching is saturated by JNK3 CS and blocked by pseudo-palmitoylated JNK3 (Parlm). Representative Wnt7a non-treated or treated hippocampal neurons transfected with indicated constructs are shown. Scale bar: 10 μm. (d) Differential effects of JNK3 CS and JNK3 Parlm on axonal branching of hippocampal neurons induced by Wnt7a. After Wnt7a induction, DsRed2 alone, JNK3 WT and JNK3 CS groups show similar numbers of total axonal branches (DsRed2 alone+Wnt7a/red, 9.1±0.5, n=62; WT+Wnt7a/green, 9.5±0.7, n=41; CS+Wnt7a/purple, 10.3±0.6, n=46; F=1.3, P=0.28) and total axonal length (fold change to DsRed2 alone, DsRed2 alone+Wnt7a, 1.5±0.4; WT, 1.6±0.8; CS, 1.6±0.6; F=0.34, P=0.71). The JNK3 Parlm group shows fewer axonal branches (4.3±0.3, n=56; F=39.78, P<0.01) and reduced axonal length (fold change, 0.9±0.4; F=19.14, P<0.01) compared with other Wnt7a-treated groups. Quantifications show mean±S.E.M. One-way ANOVA test. ^*P<0.05 and ^**P<0.01, respectively. (e) Pseudo-palmitoylated JNK3 inhibits normal axonal development. Hippocampal neurons transfected with JNK3 Parlm develop fewer axonal branches (DsRed2 alone/black, 5.0±0.2; Parlm/gray, 3.7±0.3, n=33) and shorter axons (fold change to DsRed2 alone, Parlm, 0.8±0.1). t-test. Scale bar: 10 μm