Fig 2: JIP1 is a substrate of Cdk5. A Bacterially expressed GST-tagged JIP1 was subjected to in vitro kinase assay by Cdk5/p25 and 1 µCi of [?-32P] ATP with or without Cdk5 inhibitor Roscovitine (Rosco) at 30°C for 30 min. The proteins were separated by SDS-PAGE for autoradiography. B, C Recombinant His-JIP1 was incubated with Cdk5/p25 with or without 20 µM of Roscovitine for the indicated duration. The proteins were subjected to western blot with anti-phospho-Serine (p-Ser) (B) or anti-phospho-Threonine (p-Thr). * and ** indicate full length of JIP1 and truncated form of JIP1 from C-terminus, respectively (C). Data representative of three independent experiments. D Schematic of domains of JIP1. Alignment of JIP1 protein sequence from different species analyzed by ClustaIW2 to show three conserved sites optimal for Cdk5 phosphorylation. E–G Identification of Cdk5 phosphorylation sites in JIP1. Ser197, Thr 205, and Ser 235 were all replaced with Ala. An in vitro kinase assay was carried out with purified active Cdk5/p35 incubated with WT-JIP1 (JIP1) or its Ala mutants, S197A (E), T205A (F), and S235A (G) and examined by western blot analysis using anti-phospho-Serine (p-Ser) or anti-phospho-Threonine (p-Thr). Data representative of three independent experiments. H Total cell lysate from p35 WT and KO neurons at DIV 3 was subjected to western blot analysis with custom pJIP1@T205 antibody (bands at ~100 and ~120 kDa, pJIP1@T205). Both bands from p35 WT or KO neurons were used for the measurement of JIP1 phosphorylation by Cdk5. I Quantification of phospho-JIP1 T205 levels relative to p35 WT neurons. WT, n=8 and KO, n=5, “n” equals the number of animals. Student’s t-test was used for statistical analysis and the data are presented as mean ± SEM. **** p < 0.0001

Fig 3: p35 interacts with JIP1. A Identification of JIP1 as p35-binding protein. Four peptides were identified and matched to mouse JIP1 by tandem mass spectrometry. B One microgram of bacterially expressed GST or GST fused-JIP1 was incubated with 2 μg His-p35 at 4°C for 2 h. The GST-tagged proteins were retrieved with GSH beads. The proteins were subjected to SDS-PAGE and detected using anti-p35 antibody by western blot. Amido black was used to visualize protein levels. C Endogenous p35 interacts with JIP1 in brain lysate. One microgram of IgG as control or JIP1 antibody was incubated with 500 μg of mouse brain lysate. Antibodies were pulled down using 50 μl goat-anti-rabbit IgG beads and the coupled proteins were subjected to SDS-PAGE followed by western blotting with anti-p35 antibody. D Confirmation of the specific interaction between p35 and JIP1 using p35 KO cortical neurons. Control IgG or anti-p35 was incubated with lysates from p35 WT or KO cortical neurons. The IPed proteins were immunoblotted with anti-JIP1 antibody. E Two-microgram control IgG or anti-p35 was incubated with lysate from JIP1 WT and KO neurons and the IPed proteins were immunoblotted with anti-JIP1 antibody. Images are representative of at least three independent experiments

Fig 4: Phosphorylated JIP1 rescues the defect in axonal length in JIP1 KO and p35 KO neurons. A Representative images of JIP1 WT and KO neurons expressing GFP, T205A, T205D, and JIP1. Axon length of GFP positive neurons was analyzed 3 days after the infection. Scale bar, 50 µm. B Quantification of axon length relative to GFP control analyzed by two-way ANOVA. Data presented as mean ± SEM; n=233 for GFP, n=230 for T205A, n=231 for T205D, and n=198 for JIP1 for JIP1 WT neurons, and n=224 for GFP, n=227 for T205A, n=97 for T205D, and n=151 for JIP1 for JIP1 KO neurons. “n” represents the number of neurons from one experiment. **** indicates p < 0.0001. C Representative images of axonal length of p35 KO or WT neurons expressing GFP, T205A, T205D, and JIP1 3 days after infection. Scale bar, 50 µm. D Quantification of axonal length relative to GFP control analyzed by two-way ANOVA. Data presented as mean ± SEM; n=87 for GFP, n=85 for T205A, n=81 for T205D, and n=72 for JIP1 for p35 WT neurons, and n=138 for GFP, n=114 for T205A, n=72 for T205D, and n=116 for JIP1 for p35 KO neurons. “n” equals the number of neurons from one experiment and representative of 3 independent experiment. *** p < 0.001 and **** p < 0.0001. E Quantification of axon length in E14.5 embryonic brains subjected to in utero electroporation with GFP and T205D plasmids. Brains were collected and stained with anti-GFP for the measurement of axonal length 3 days after electroporation. Data presented as mean ± SEM. Student’s t-test was used to compare groups of data; n=3 for GFP and T205D for JIP1 WT brains, and n=3 for GFP and n=4 for T205D for JIP1 KO brains. “n” equals the numbers of animals and > 50 GFP-positive cells from each animal were used for quantification. * p < 0.05

Fig 5: p35 degradation in neurons is mediated through the E3 ubiquitin ligase PJA2.(a) GSNO treatment increased p35 polyubiquitination level. Cultured rat cortical neurons were treated with GSNO in the presence of the proteasome inhibitor MG132. The protein lysate was subjected to immunoprecipitation using p35 antibody followed by western blot analysis for polyubiquitin. (b) p35 associated with PJA2 in the mouse hippocampus. Hippocampal lysates of 3-month-old mice were immunoprecipitated (IP) using p35 or PJA2 antibodies (IgG as control) as indicated, and the proteins in the co-immunoprecipitates were examined by western blot analysis. (c,d) p35 degradation is mediated by the E3 ligase PJA2. (c) PJA2 was knocked down in cultured rat cortical neurons, and the endogenous expression of p35 was examined by western blot analysis. (d) Quantification of p35 protein level (relative to the actin level). Data are normalized to the control and represent the mean±s.e.m. of four independent experiments. *P=0.016, **P=0.0018; one-way analysis of variance (ANOVA) with the Student–Newman–Keuls test. (e,f) GSNO promotes p35 degradation in a PJA2-dependent manner. (e) PJA2 was knocked down with PJA2-shRNA (#1) in cultured rat cortical neurons for 4 days, the cells were treated with GSH/GSNO for 1 h and p35 protein level was examined by western blot analysis. (f) Quantification of p35 level (relative to the actin level). Data are normalized to control group and represent the mean±s.e.m. of six independent experiments. ***P=6.81E−7, ###P=6.42E−5; one-way ANOVA with the Student–Newman–Keuls test.