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1 Supplementary Materials for JNK3 Couples the Neuronal Stress Response to Inhibition of Secretory Trafficking Guang Yang,* Xun Zhou, Jingyan Zhu, Rui Liu, Si Zhang, Ainsley Coquinco, Yongting Chen, Yanhua Wen, Luba Kojic, William Jia, Max S. Cynader* *Corresponding author. (M.S.C.); (G.Y.) Published 9 July 2013, Sci. Signal. 6, ra57 (2013) DOI: /scisignal The PDF file includes: Fig. S1. Subcellular localization of JNK3 induced by PATs. Fig. S2. Kinase activity independent localization of JNK3 to the Golgi complex. Fig. S3. Enhanced phosphorylation of palmitoylated JNK3. Fig. S4. Inhibition of VSVG trafficking by palmitoylated JNK3. Fig. S5. Inhibition of vesicle secretion from the Golgi by palmitoylated JNK3. Fig. S6. Reduction of surface GluR1 induced by NMDA excitotoxicity. Fig. S7. Disruption of secretory transport system and depletion of PI4P induced by palmitoylated JNK3. Fig. S8. Reduction of neuronal surface GluR1 induced by Sac1. Fig. S9. Interaction between Sac1 and palmitoylated JNK isoforms. Fig. S10. Identification of two JNK3-binding motifs on Sac1. Fig. S11. Identification of potential Sac1-binding motifs on JNK3. Fig. S12. Enlargement of the Golgi-resident pool of PI4P in neurons by Sac1 knockdown. Fig. S13. Disruption of the Sac1-JNK3 interaction by synthetic blocking peptides.

2 Supplementary Materials Fig. S1. Subcellular localization of JNK3 induced by PATs. (A) Western blot analysis of palmitoylation of JNK3 in rat cortical neurons assessed by the fatty acyl exchange assay. PD, pulldown; IB, immunoblotting; IP: immunoprecipitation; HAM, hydroxylamine. (B) GFP- JNK3 was transfected into COS7 cells together with individual zdhhc PATs (red) as indicated by number. Images that show translocation of JNK3 to the perinuclear region induced by the PAT are underlined. (C) Wild-type JNK3 (WT) was expressed in COS7 cells with individual PATs as indicated, and profiles of JNK3 distribution along the dotted lines are shown on the right. The shaded box represents the perinuclear region. (D) Western blot analysis

3 of palmitoylated JNK3 without (control) or with the indicated PATs in HEK293 cells. Bar graph shows the means and standard error of the fold-change of palmitoylation of JNK3 in the presence of individual PATs compared with the JNK3-alone control (n=3 experiments). ** p<0.01, ANOVA with Tukey test.

4 Fig. S2. Kinase activity independent localization of JNK3 to the Golgi complex. (A) Colocalization of transiently expressed GFP-JNK3 Parlm or KD-Parlm with GM130 (red), a marker of the Golgi complex in COS7 cells. Small vesicles containing JNK3 Parlm and KD- Parlm are indicated by arrows. Profiles of subcellular distribution of JNK3 (green line) and GM130 (red line) along the dotted lines (intensity versus distance) are shown at the bottom. (B) Western blot analysis of JNK3 variants at the Golgi assessed by gradient fractionation. HEK293 cells were transfected with wild-type JNK3 (WT) or JNK3 mutants (CS or Parlm) with or without zd17. Fractions 3 to 5 (dotted box) enriched with GM130 represent Golgi contents. Representative figures from three independent experiments are shown. (C) COS7 cells were transfected with GFP-JNK3 and zd17. GFP-JNK3 and GM130 (red) was examined in cells treated with BFA (5 µm) for 30 minutes, or 4 hours after BFA washout (right top panel). Colocalization of JNK3 with GM130 is indicated by arrows. Cells transfected with GFP-JNK3

5 and zd17 were examined with aggresome markers HSP70 (red) and vimentin (red) (right panel). Arrows indicate the corresponding locations of JNK3. Scale bars: 10 µm. (D) Kinase activity of the kinase-deficient mutant JNK3 (KD) relative to wild-type JNK3 (WT) expressed in HEK293 cells. The kinase assay measured phosphorylation at Ser 63 of purified c-jun protein. All representative figures and quantifications were from three independent experiments. ** p<0.01, Two-tailed t-test.

6 Fig. S3. Enhanced phosphorylation of palmitoylated JNK3. (A) Interaction between zd17 and wild-type JNK3 (WT) or the JNK3-CS mutant in transfected HEK293 cells was examined by coimmunoprecipitation. (B) The phosphorylation of wild-type or mutant (CS) JNK3 at Thr 183 /Tyr 185 in the absence or presence of zd17 in HEK293 cells. (C) The phosphorylation of JNK3 in the

7 presence of zd15 in transfected HEK293 cells was assessed after treatment with 400 mm sorbitol (30 min) to induce osmotic stress. (D) The phosphorylation status of JNK3 WT and the Parlm mutant was assessed in transfected HEK293 cells. (E) Immunofluorescent detection of the phosphorylation (red) of GFP-tagged wild-type or mutant (CS or Parlm) JNK3 transfected with or without zd15 in HEK293 cells. Arrows and hollow arrows indicate the presence and the absence of phosphorylation signal, respectively. Scale bars: 10 µm. All images and bar graphs are representative of and quantified from three independent experiments. ** p<0.01, Two-tailed t- test.

8 Fig. S4. Inhibition of VSVG trafficking by palmitoylated JNK3. (A) VSVG- mcherry (red), myc-zd17 and GFP-JNK3 variants were expressed in cultured rat hippocampal neurons as indicated. Accumulation and colocalization of VSVG and JNK3 at the Golgi complex are indicated by arrows. (B) VSVG- mcherry (red) was coexpressed with GFP-JNK3 variants and myc-zd17 in COS7 cells as indicated. Distribution profiles of JNK3 (green line) and VSVGmcherry (red line) along the dotted line are shown at the bottom. (C) Transport velocity of VSVG-ts045 (green) to the plasma membrane of COS7 cells transfected with indicated constructs was assessed after shifting cells from 39.5 C to 32 C. Line graph shows the means of the data values for two independent experiments with bars representing the range of the original values from two experiments. Scale bars: 10 µm.

9 Fig. S5. Inhibition of vesicle secretion from the Golgi by palmitoylated JNK3. (A) GFP- NCAM, GFP-p75, or GFP-BDNF was coexpressed with DsRed2-tagged JNK3 variants and myc-zd17 in COS7 cells. Cells expressing either DsRed2-JNK3 with NCAM or only NCAM are indicated by arrows or an arrowhead, respectively. (B) The distribution of HA-GluR1 (red) was examined in COS7 cells expressing GFP-JNK3 variants and myc-zd17 (blue). (C) Localization of wild-type GFP-GluR1 (GluR1 WT ) or the C585S mutant (GluR1 C585S ) was examined in COS7 cells expressing either GFP-JNK3 WT or the CS mutant together with myc-zd17 (red). Scale bars: 10 µm. Images are representative of three experiments.

10 Fig. S6. Reduction of surface GluR1 induced by NMDA excitotoxicity. (A) Detection of GluR1 and the synaptic scaffolding protein PSD-95 (postsynaptic density protein 95) along dendrites in rat hippocampal neurons using either non-permeable (Surface) or permeable (Total) immunofluorescence methods. Surface GluR1 on dendrites in the dotted boxes are magnified below each image (X). Images are representative of two experiments. (B) NMDA induced a steady reduction of surface GluR1 in cultured rat cortical neurons in the presence of the JNK inhibitor SP (10 µm). Line graph shows the means and standard error relative to the untreated control (at 2 hours, n=14 cells; at 4 hours, n=11 cells, at 16 hours, n=14 cell; at 24 hours, n=9 cells). (C) Western blot analysis (IB) of surface or total GluR1 from rat cortical neurons 16 hours after treatment with NMDA in the presence of SP or NIMoE (1 µm). Bar graphs show the means and standard error in treated neurons relative to untreated controls (n=3 experiments). ** p<0.01, Two-tailed t-test with Bonferroni correction. Scale bars: 10 µm.

11 Fig. S7. Disruption of secretory transport system and depletion of PI4P induced by palmitoylated JNK3. (A) COS7 cells expressing GFP-JNK3 and myc-zd17 were stained with selective markers (red) as indicated across the top. Cells expressing or not expressing JNK3 and zd17 are indicated by arrows or arrowheads, respectively. (B) Golgi-resident PI4P was labeled in COS7 cells by both Fapp1-GFP (green) and an antibody against PI4P (red). (C) PI4P in COS7 cells expressing DsRed2-tagged JNK3 variants (red) and myc-zd17 was examined with Fapp1- GFP (green). The intensity profiles (control, blue; KD+zD17, red; KD-Parlm, green) of Fapp1- GFP along the dotted lines are shown to the right. The shaded box represents normal background Fapp1- GFP. (D) PI4P in COS7 cells expressing GFP-tagged JNK3 variants (green) and myczd17 was examined with an antibody against PI4P (red). The intensity profiles were measured as in (C). Scale bars: 10 µm. All data is representative of three experiments.

12 Fig. S8. Reduction of neuronal surface GluR1 induced by Sac1. (A) PI4P in rat hippocampal neurons transiently expressing FLAG-Sac1 WT, LZ or K2a (red) was assessed by Fapp1-GFP (green). Accumulation of Fapp1-GFP at the Golgi is indicated by arrows. Bar graph shows the means and standard error of the intensity of Fapp1-GFP at the Golgi complex (n=30 cells). (B) Surface GluR1 was examined in rat hippocampal neurons transiently expressing GFP-tagged WT, LZ or K2a Sac1. Bar graph shows the means and standard error of the percentage of the density of surface GluR1 in neurons expressing GFP-Sac1 variants compared to the GFP-alone control (n=18 cells). * p<0.05, ** p<0.01, ANOVA with Tukey test. Scale bars: 10 µm.

13 Fig S9. Interaction between Sac1 and palmitoylated JNK isoforms. (A) Coimmunoprecipitation and western blot analysis of the interaction between Sac1 and JNK isoforms in the presence or absence of zd17 (as indicated) in HEK293 cells. Bar graph shows the means and standard error of the percentage of the strength of the Sac1 interaction with JNK isoforms compared to the Sac1-JNK1 interaction (control) (n=3 experiments). ** p<0.01, Two-tailed t-test with Bonferroni correction; (B) COS7 cells transfected with zd17 and GFP-JNK isoforms (green) as indicated were assessed for colocalization with Golgi marker GM130 (red). Accumulation or absence of JNK isoforms at the Golgi complex is indicated by arrows or arrowheads respectively. Images are representative of three experiments. Scale bar: 5 µm.

14

15 Fig. S10. Identification of two JNK3-binding motifs on Sac1. (A) Analysis of the JNK3-Sac1 interaction using an in vitro binding assay. (B) Co-immunoprecipitation and western blot analysis of the interaction between JNK3 and FLAG-tagged wild-type or mutant Sac1 m139-del (motif m139 deleted), m412-del (motif m412 deleted) or m139,412-del (both motifs deleted) in HEK293 cells. Bar graph shows the means and standard error of the percentage of the strength of the GFP- JNK3 interaction with mutant FLAG-Sac1 compared to the wild-type FLAG-Sac1 control (n=3 experiments). ** p<0.01, Two-tailed t-test. (C) Protein sequences of yeast (CAA82057) and rat (NP_446250) Sac1 were aligned and the regions corresponding to motif 139 and motif 412 are marked in blue for yeast Sac1 and yellow for rat Sac1. (D) The 3D structure of yeast Sac1 fragment is from Protein Data Bank (accession number 3LWT). These two motifs (139 and 412) locate at the loop regions that are present at the surface of the proteins and are separated by a betasheet structure corresponding to region aa (E) Analysis of sequence similarity across rat non-redundant protein sequence database with motifs m139 and m412.

16 Fig. S11. Identification of potential Sac1-binding motifs on JNK3. (A) Screening of potential binding sites on a peptide array of human JNK3a2. The peptide array was incubated with in vitro with purified human Sac1 proteins, and blotted with the anti-sac1 antibody. Four clusters of spots (A, B, C, D) on the membrane were consistently identified as strongly positive in two repeats of the assay. (B) The full sequences of these positive peptide spots cover two loose regions on JNK3, as labeled in yellow shades. Based on the binding efficiency detected on the peptide array, three potent Sac1-Binding Motifs (SBM-1, 2, and 3) were marked in red squares. The sequences of these SBMs are highly conserved among JNK isoforms. The dual-phosphorylation motifs (Thr-Pro-Tyr) on JNKs are marked in red. (C) Identification of SBM2 as the key motif for Sac1 interaction. GFP- JNK3 mutants were generated by deleting corresponding motifs (SBM1, 2, and 3) individually (SBM1-del, SBM2-del, or SBM3-del) or simultaneously (SBMall-del). The interaction between Sac1 and these JNK3 mutants were examined by co-immunoprecipitation in lysates from HEK293

17 cells. Representative figures and quantifications from three independent experiments are shown. ** p<0.01, Two-tailed t-test with Bonferroni correction. (D) The structural organization of SBM2 is shown (Protein Data Bank, 3OY1).

18 Fig. S12. Enlargement of the Golgi-resident pool of PI4P in neurons by Sac1 knockdown. (A) Western blot analysis of knockdown of myc-flag-tagged rat Sac1 (NM_053798) in HEK293 cells by shrna (sir1 or sir2). Bar graph shows the means and standard error of the percentage of the amount of FLAG-Sac1 in cells expressing Sac1 shrnas compared to the psuper-expressing control (n=4 experiments). (B) Western blot analysis of knockdown of endogenous Sac1 in PC12 cells by shrna (sir1 or sir2). Bar graph shows the means and standard error of the percentage of the amount of Sac1 in cells expressing Sac1 shrnas compared to the psuper-expressing control (n=3 experiments). (C) PI4P was detected by Fapp1-GFP in rat hippocampal neurons expressing the psuper control or Sac1 shrnas for 48 hours. The enrichment of PI4P in the Golgi is indicated by arrows. Bar graph shows the means and standard error of the intensity of Fapp1-GFP at the Golgi (psuper, n=6 cells; Sac1-siR2, n=5 cells). Scale bars 10 µm. For all data, * p<0.05, ** p<0.01, Two-tailed t-test with Bonferroni correction.

19 Fig. S13. Disruption of the Sac1-JNK3 interaction by synthetic blocking peptides. (A) The endocytosis of TAT-sequence fused, FITC labeled NIMoE peptides (pep130 and pep412) in COS7 cells was examined at indicated time points. Small endocytosis vesicles are indicated by arrows. (B) Western blot analysis of the effect of pep130 and pep412 individually or in combination on the JNK3-Sac1 interaction. Peptides (5 µm) were treated to rat cortical neurons for 16 hours and the interaction between JNK3 and Sac1 were assessed by co-immunoprecipitation and quantified as mean and standard error of the percentage of the interaction between JNK3 and Sac1 in the presence of peptides compared to the untreated control: pep139 only, 111.4±5.1%, p=0.18; pep412 only, 97.5±22.3, p=1.82; pep139 and 412, 44.3±7.1%, p=0.003; Two-tailed t-test with Bonferroni correction; n=3 experiments. (C) Surface GluR1 was examined in rat hippocampal neurons treated with NMDA in the presence or absence of individual blocking peptides (pep139 or pep412). Bar graph shows the means and standard error of the percentage of surface GluR1 in treated neurons

20 compared to the untreated control (n=5 cells). Two-tailed t-test with Bonferroni correction. Scale bars: 10 µm.

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