The palmitoyl acyltransferases ZDHHC5 and ZDHHC8 are uniquely present in DRG axons and control retrograde signaling via the Gp130/JAK/STAT3 pathway

Abstract

Palmitoylation, the modification of proteins with the lipid palmitate, is a key regulator of protein targeting and trafficking. However, knowledge of the roles of specific palmitoyl acyltransferases (PATs), which catalyze palmitoylation, is incomplete. For example, little is known about which PATs are present in neuronal axons, although long-distance trafficking of palmitoyl-proteins is important for axon integrity and for axon-to-soma retrograde signaling, a process critical for axon development and for responses to injury. Identifying axonally targeted PATs might thus provide insights into multiple aspects of axonal biology. We therefore comprehensively determined the subcellular distribution of mammalian PATs in dorsal root ganglion (DRG) neurons and, strikingly, found that only two PATs, ZDHHC5 and ZDHHC8, were enriched in DRG axons. Signals via the Gp130/JAK/STAT3 and DLK/JNK pathways are important for axonal injury responses, and we found that ZDHHC5 and ZDHHC8 were required for Gp130/JAK/STAT3, but not DLK/JNK, axon-to-soma signaling. ZDHHC5 and ZDHHC8 robustly palmitoylated Gp130 in cotransfected nonneuronal cells, supporting the possibility that Gp130 is a direct ZDHHC5/8 substrate. In DRG neurons, Zdhhc5/8 shRNA knockdown reduced Gp130 palmitoylation and even more markedly reduced Gp130 surface expression, potentially explaining the importance of these PATs for Gp130-dependent signaling. Together, these findings provide new insights into the subcellular distribution and roles of specific PATs and reveal a novel mechanism by which palmitoylation controls axonal retrograde signaling.

Keywords: DLK; DRG; axon; intracellular trafficking; neuron; protein acylation; protein kinase; protein palmitoylation.

Figure 1.

ZDHHC5 and ZDHHC8 are preferentially targeted to DRG axons compared with all other PATs. A, images of HA fluorescent signal (left panels) and merged HA, GFP, and DAPI (blue, to detect nuclei) signals (right panels) from individual cultured DRG neurons co-infected with control lentivirus expressing GFP plus a second virus expressing the indicated HA-tagged ZDHHC-PAT. PAT numbering reflects updated gene names (2). Scale bar, 20 μm. B, quantified axon/soma fluorescence intensity ratio for each of the indicated PATs, normalized to GFP. Blue dotted line, mean axon/soma fluorescence intensity ratio for all PATs from all determinations. One-way ANOVA was used with the Bonferroni post hoc test (F(23, 220) = 9.332, p < 0.0001); ****, p < 0.0001; **, p < 0.01, compared with average axon/soma ratio for all PATs from all determinations. n = 7–13 neurons/condition. Bars, mean ± S.D. (error bars).

Figure 2.

Endogenous ZDHHC5 localizes to axons in DRG neurons. A, cultured DRG neurons were infected with either control virus or virus expressing shRNA against ZDHHC5 (ZDHHC5sh). Zdhhc5 knockdown was verified by immunoblotting lysates of infected neurons. Molecular weight markers (Mw (kDA)) are indicated on the left-hand side of this and all subsequent blots. The efficacy of ZDHHC5 knockdown in this and replicate experiments is quantified in Fig. S2. B, DRG neurons were cultured in microfluidic chambers and proximal chambers (containing cell somas and proximal axons (Soma & axon)) and distal chambers (containing distal axons but no neuronal cell bodies (Axon)) were lysed separately in denaturing buffer. Equivalent volumes of each fraction were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. Endogenous ZDHHC5 is clearly detected in distal axon fractions. Right panel plot, quantified ZDHHC5 in the indicated microfluidic compartments, relative to tubulin (n = 4 determinations). C, images of DRG neurons infected with lentivirus expressing GFP with or without Zdhhc5 shRNA, following fixation and immunostaining with the indicated antibodies. Zddhc5 knockdown greatly reduces axonal signal recognized by the anti-ZDHHC5 antibody (quantified in Fig. S2). Scale bar, 20 μm. Bars, mean ± S.D. (error bars).

Figure 3.

ZDHHC5/8 are critical for retrograde signaling by the Gp130/JAK/STAT3 pathway, but not the DLK/JNK pathway. A, DRG neurons cultured in microfluidic chambers were infected with the indicated viruses. Cultures were left untreated, or distal axons were axotomized by aspiration. Cell body chambers were then immunostained to detect phospho-c-Jun (p-c-Jun) and DAPI. B, quantified data from A reveal that axotomy-induced c-Jun phosphorylation, which is strongly DLK-dependent (28), is not affected by Zdhhc5/8 knockdown. ns, not significantly different from control-infected axotomized condition, two-way ANOVA: virus p = 0.2754 (F(1, 18) = 1.265), axotomy p < 0.0001 (F(1, 18 = 27.89), interaction p = 0.3892 (F(1, 18) = 0.7785), n = 5–6 determinations/condition. C, distal axonal chambers of DRG neurons cultured as in A were treated with or without 10 ng/ml rat CNTF, and cell body chambers were fixed and immunostained to detect STAT3 phosphorylation (pSTAT3) and DAPI. D, quantified data from C reveal that Zdhhc5/8 knockdown significantly reduces axotomy-induced STAT3 phosphorylation. n = 4 determinations/condition. **, p < 0.01, two-way ANOVA: virus p = 0.0119 (F(1, 12) = 8.766), treatment p = 0.0003 (F (1, 12) = 25.27), interaction p = 0.0166 (F(1, 12) = 7.74). E, Western blots of lysates from conventionally cultured DRG neurons (sister cultures of those used for microfluidic experiments in A–D) infected with the indicated viruses confirm effective reduction of ZDHHC5 and ZDHHC8 protein levels by their respective shRNAs (see also quantified data in Fig. S2). Bars, mean ± S.D. (error bars).

Figure 4.

Gp130 is palmitoylated in a ZDHHC5/8-dependent manner. A, HEK293T cells were transfected with an empty vector or with Gp130-expressing vector and subsequently treated with palmitoylation inhibitor 2-bromopalmitate (2BP) or vehicle. ABE fractions were prepared and blotted to detect palmitoyl-Gp130 (top). Lysates were blotted to detect total Gp130 expression (bottom). The image in the top panel is from a single Western blot analysis, cropped to remove intervening spacer lanes. The experiment shown is representative of three individual determinations. B, HEK293T cells were transfected with Gp130 cDNA plus the indicated ZDHHC5 or ZDHHS5 cDNAs and ABE assays performed. ABE fractions were blotted to detect palmitoyl-Gp130 (top). Lysates were blotted to detect total Gp130 and total ZDHHC5 expression (middle and bottom panels). C, quantified data for palmitoylated-to-total (Palm/total) Gp130 from B confirm that ZDHHC5 significantly increases Gp130 palmitoylation, whereas catalytically inactive ZDHHS5 does not. *, p < 0.05, nonparametric one-way ANOVA with Dunn's multiple-comparison post hoc test (p = 0.0086, Kruskal–Wallis statistic = 7.848), 4–5 determinations/condition. D, as in B, except that cells were transfected with the indicated ZDHHC8 or ZDHHS8 cDNAs, and lysates were blotted with Gp130 and ZDHHC8 antibodies. E, quantified data from D confirm that ZDHHC8 significantly increases palmitoyl-Gp130 levels, whereas catalytically inactive ZDHHS8 does not. *, p < 0.05, nonparametric one-way ANOVA with Dunn's multiple-comparison post hoc test (p = 0.0086, Kruskal–Wallis statistic = 7.848), 4–5 determinations per condition. F, cultured sensory neurons were infected with control virus or with viruses expressing ZDHHC5 and ZDHHC8 shRNAs. ABE fractions were prepared from lysates and blotted to detect palmitoyl-Gp130 and -GAP-43 (left panels). Lysates were blotted to detect total ZDHHC5, ZDHHC8, Gp130, GAP-43, and tubulin expression (right panels). G, quantified data from n = 4 determinations from F confirm that Zdhhc5/8 knockdown significantly reduces Gp130 palmitoylation. *, p < 0.05, t test. H, quantified data from n = 4 determinations from F confirm that Zdhhc5/8 knockdown does not significantly reduce Gp130 total levels. n.s., not significant, t test. I, quantified data from n = 4 determinations from F confirm that Zdhhc5/8 knockdown does not significantly reduce Gp130 palmitoylation, when normalized to total Gp130. n.s., not significant, t test. Bars, mean ± S.D. (error bars).

Figure 5.

ZDHHC5/8 control Gp130 surface expression. A, surface fractions (left column) and total lysates from cultured DRG neurons infected with the indicated viruses were immunoblotted with the indicated antibodies. B, quantified data from A reveal that Zdhhc5/8 knockdown significantly reduces surface/total Gp130 (left histogram; *, p < 0.05, t test) but does not affect surface/total levels of the axonal palmitoyl-protein neurofascin (right histogram; n.s., nonsignificant, t test, n = 8 and n = 4 determinations/condition, respectively). Bars, mean ± S.D. (error bars).