Journal: Nature Communications

Article Title: Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

doi: 10.1038/s41467-017-01609-x

Figure Lengend Snippet: p38 MAPK mediates ER stress-induced CMA. a ER stress activates p38α MAPK. Phospho-p38α MAPK (p-p38α) levels were increased in SN4741 cells after exposure to Tg or Tu for 1 h or 3 h (left and middle sets of panels). Tg (3 h) increased the level of p38 MAPK substrate p-ATF2 (activating transcription factor 2) in SN4741 cells and Tg (12 h) decreased the level of MEF2D (right set of panels). b Inhibition of p38 MAPK prevents ER stress-induced CMA activation. CMA activity was determined by lysosome binding (top left) and uptake (bottom left) assays using a commercially purified CMA substrate RNase A following incubation of lysosomes purified SN4741 cells exposed to Tg with or without SB203580 for 12 h. SN4741 cells were transfected with dominant-negative p38α MAPK (DN-p38α) or control vector, treated with Tg as described above, and assayed for RNase A binding and uptake (right top and middle panels). Bottom shows the levels of LAMP2A in lysosomes under various conditions. c Increasing p38 MAPK activity is sufficient to activate CMA. Lysosomes isolated from SN4741 cells transfected with constructs as indicated were blotted for LAMP2A and Hsc70 (left) and tested for RNase A uptake (right). d ER stress increases the level of p-p38α MAPK on lysosomes. Cytosolic fraction (CL) and purified lysosomes (Ly) isolated from SN4741 after Tg treatment (0.3 μM, 2 h) were blotted for various proteins as indicated (left panel: LAMP1, lysosome marker; PDI, ER marker; cytochrome C (Cyto C), mitochondrial marker) or were analyzed by immunocytochemistry (right panel. Scale bar = 10 µm). e SB203580 attenuates ER stress-induced increase of LAMP2A on lysosomal membrane. SN4741 cells were exposed to Tg for 12 h with or without 1 h SB203580 pretreatment. Lysosomal membrane and matrix fractions were analyzed for LAMP2A (left). Purified lysosomes were blotted for oligomerized LAMP2A (right). Quantifications are shown for a , b , c , and e ( n = 3 (ANOVA with Turkey test); ** p < 0.005 vs. control and ## p < 0.005 vs. corresponding challenge alone)

Article Snippet: The chemicals and their sources are as follows: SB203580 (cat#5633) from Cell Signaling; 4μ8C (IRE1 inhibitor III, cat#412512), cycloheximide (cat#239763), geldanamycin (cat#345805), and GSK2606414 (PERK inhibitor I, cat#516535) from EMD Millipore; 32 P-ATP (cat#50-905-0808) from Perkin Elmer; 3-methyladenine (3MA, cat#M9281), 6-aminonicotinamide (6-AN, cat#A68203), 6-hydroxydopamine (6-OHDA, cat#H4381), ammonium chloride (NH 4 Cl, Cat#254134), ATP (cat#A2383), BFA (#B7651), DAPI (cat#D9542), MG132 (cat#M7449), Tg (cat#T9033), Tu (cat#654380), and β-mercaptoethanol (cat#M6250) from Sigma-Aldrich; and Hoechst 33258 (cat#H3569) from Thermo Fisher.

Techniques: Inhibition, Activation Assay, Activity Assay, Binding Assay, Purification, Incubation, Transfection, Dominant Negative Mutation, Control, Plasmid Preparation, Isolation, Construct, Marker, Immunocytochemistry, Membrane

Journal: Nature Communications

Article Title: Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

doi: 10.1038/s41467-017-01609-x

Figure Lengend Snippet: p38 MAPK directly phosphorylates LAMP2A. a High level of p38 MAPK leads to LAMP2A phosphorylation in SN4741 cells. SN4741 cells were transfected with p38 MAPK for 40 h with or without SB203580 (20 μM), and proteins were collected for IP with anti-LAMP2A and IB with antibodies to p-Thr-substrates, p-Ser-substrates, or LAMP2A. b p38 MAPK directly phosphorylates human LAMP2A in vitro. Endogenous LAMP2A proteins were immunoprecipitated from HEK293 cells, and the precipitates were incubated with purified p38 MAPK in vitro kinase assay with 32 P-ATP or cold ATP following by anti-p-Thr or p-Ser blotting. ( c ) T211 and T213 are required for p38 MAPK-mediated phosphorylation of LAMP2A. HEK293 cells were transfected with myc-LAMP2A (wt) or (mt, T211A and T213A) for 40 h, and proteins were collected for IP with anti-myc for p38 MAPK in vitro kinase assay (top left); and SN4741 cells were transfected with LAMP2A (wt), or LAMP2A(mt) with Flag-p38 MAPK for 40 h and proteins were collected for IP with anti-myc and IB with anti-p-Thr-substrate antibody. d ER stress induces LAMP2A phosphorylation in a p38 MAPK-dependent manner. SN4741 cells were treated with Tg (0.3 μM) or Tu (3 μg/ml) for 2 or 1 h, respectively. Total (top) or lysosomal (bottom) proteins were extracted from the cells for IP with LAMP2A antibody and IB with anti-p-Thr-substrate antibody. e ER stress increases lysosomal LAMP2A and its oligomerization in a phosphorylation-dependent manner. SN4741 cells were transfected with myc-wt-LAMP2A or myc-mt-LAMP2A (T211A and T213A) for 24 h and then were treated with Tg (0.3 μM) or Tu (3 μg/ml) for 12 h. Lysosomal proteins were blotted following SDS-PAGE (top) or blue negative gel (bottom) with anti-myc antibody. f ER stress increases CMA activity in an LAMP2A phosphorylation-dependent manner. SN4741 cells were transfected with pcDNA3, wt-LAMP2A, or mt-LAMP2A (T211A and T213A) for 24 h and then were treated with Tg (0.3 μM) or Tu (3 μg/ml) for 12 h. Cytoplasmic proteins were blotted with an MEF2D antibody (top), and intact lysosomes were assessed in uptake assay (bottom). Quantifications are shown for a , c , d , e , and f ( n = 3. All values are mean ± s.d. (ANOVA with Turkey test). ** p < 0.005 vs. control and ## p < 0.005 vs. corresponding challenge alone)

Article Snippet: The chemicals and their sources are as follows: SB203580 (cat#5633) from Cell Signaling; 4μ8C (IRE1 inhibitor III, cat#412512), cycloheximide (cat#239763), geldanamycin (cat#345805), and GSK2606414 (PERK inhibitor I, cat#516535) from EMD Millipore; 32 P-ATP (cat#50-905-0808) from Perkin Elmer; 3-methyladenine (3MA, cat#M9281), 6-aminonicotinamide (6-AN, cat#A68203), 6-hydroxydopamine (6-OHDA, cat#H4381), ammonium chloride (NH 4 Cl, Cat#254134), ATP (cat#A2383), BFA (#B7651), DAPI (cat#D9542), MG132 (cat#M7449), Tg (cat#T9033), Tu (cat#654380), and β-mercaptoethanol (cat#M6250) from Sigma-Aldrich; and Hoechst 33258 (cat#H3569) from Thermo Fisher.

Techniques: Phospho-proteomics, Transfection, In Vitro, Immunoprecipitation, Incubation, Purification, Kinase Assay, SDS Page, Activity Assay, Control

Journal: Nature Communications

Article Title: Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

doi: 10.1038/s41467-017-01609-x

Figure Lengend Snippet: PERK-p38–MAPK–phosphor–LAMP2A pathway mediates cellular protection. a Knockdown of PERK aggravates Tg-induced cell death in SN4741 cells. SN4741 cells were transfected with si-Control and si-PERK for 48 h and then treated with Tg (1 μM) for 24 h. The cell viability was measured by WST-1 assay and data were expressed as mean ± s.e.m. ( n = 3 × 4 replicates (ANOVA with Turkey test); ** p < 0.005 vs. si-Control, and ## p < 0.005 vs. si-Control with Tg group). Total proteins were blotted for cleaved caspase 3 (top right) or PERK (bottom right). b The effect of p38 MAPK inhibition on ER stress-induced cell death is time-dependent. SN4741 cells were exposed to Tg and Tu for 3 or 6 h and then co-incubated with SB203580 for a total of 24 h Tg or Tu treatment. Cell viability was measured by WST-1 assay and data were expressed as mean ± s.e.m. ( n = 3 × 4 replicates of independent experiments (ANOVA with Turkey test); ** p < 0.005 vs. control, # p < 0.05 and ## p < 0.005 vs. Tg or Tu alone group). Right panel shows that the increase of lysosomal LAMP2A induced by Tg can be abolished by SB203580 3 h, but not 6 h post Tg stress. c LAMP2A phosphorylation is necessary for protecting cells against ER stress. SN4741 cells were transfected with pcDNA3, myc-wt-LAMP2, mt (A/A)-LAMP2A (T211A and T213A), and mt (E/D)-LAMP2A (T211E and T213D) for 24 h, exposed to Tg or Tu for another 24 h, and assessed for viability by WST-1 assay. Data were expressed as mean ± s.e.m. ( n = 3 × 4 replicates of independent experiments (ANOVA with Turkey test). * p < 0.05 or ** p < 0.005 vs. control, ## p < 0.005 vs. Tg or Tu alone group, $ p < 0.05 and $$ p < 0.01 vs. wt-LAMP2A with Tg or Tu alone group). Right panel shows the lysosomal levels of the overexpressed LAMP2A

Article Snippet: The chemicals and their sources are as follows: SB203580 (cat#5633) from Cell Signaling; 4μ8C (IRE1 inhibitor III, cat#412512), cycloheximide (cat#239763), geldanamycin (cat#345805), and GSK2606414 (PERK inhibitor I, cat#516535) from EMD Millipore; 32 P-ATP (cat#50-905-0808) from Perkin Elmer; 3-methyladenine (3MA, cat#M9281), 6-aminonicotinamide (6-AN, cat#A68203), 6-hydroxydopamine (6-OHDA, cat#H4381), ammonium chloride (NH 4 Cl, Cat#254134), ATP (cat#A2383), BFA (#B7651), DAPI (cat#D9542), MG132 (cat#M7449), Tg (cat#T9033), Tu (cat#654380), and β-mercaptoethanol (cat#M6250) from Sigma-Aldrich; and Hoechst 33258 (cat#H3569) from Thermo Fisher.

Techniques: Knockdown, Transfection, Control, WST-1 Assay, Inhibition, Incubation, Phospho-proteomics

Journal: Nature Communications

Article Title: Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

doi: 10.1038/s41467-017-01609-x

Figure Lengend Snippet: ER stress induces CMA activation in vivo. a Tu induces ER stress in mouse brain. Mice were injected (i.p.) with Tu (1 μg/g body weight), and then killed 2 days later for analysis ( n = 12). Levels of LAMP2A, Bip, and MEF2D in various brain regions of mice were determined by western blotting. b ER stress induces p-LAMP2A on striatal lysosomes in a p38 MAPK-dependent manner. Mice were treated with Tu (0.5 or 1 μg/g body weight) alone or together with SB203580 for 24 h ( n = 9). Lysosomes isolated from striatal tissue were blotted for p-p38 MAPK (Left), and for IP with LAMP2A and IB with p-Thr-substrate antibody (Right and bottom). c , d ER stress increases both LAMP2A level and CMA activity of lysosomes isolated from the striatum. Mice were injected (i.p.) as described in ( a ) with or without co-injection of SB203580 and then killed after 24 h ( n = 9). Lysosomes isolated from the striatal tissue were analyzed for LAMP2A ( c ) or analyzed for CMA activity by uptake assay ( d ). Quantifications are shown for a to d ( n = 6. All values are mean ± s.d. (unpaired two-tailed t test for LAMP2A/Bip/MEF2D, ANOVA with Turkey for others). * p < 0.05 or ** p < 0.005 vs. control and ## p < 0.005 vs. 6-OHDA)

Article Snippet: The chemicals and their sources are as follows: SB203580 (cat#5633) from Cell Signaling; 4μ8C (IRE1 inhibitor III, cat#412512), cycloheximide (cat#239763), geldanamycin (cat#345805), and GSK2606414 (PERK inhibitor I, cat#516535) from EMD Millipore; 32 P-ATP (cat#50-905-0808) from Perkin Elmer; 3-methyladenine (3MA, cat#M9281), 6-aminonicotinamide (6-AN, cat#A68203), 6-hydroxydopamine (6-OHDA, cat#H4381), ammonium chloride (NH 4 Cl, Cat#254134), ATP (cat#A2383), BFA (#B7651), DAPI (cat#D9542), MG132 (cat#M7449), Tg (cat#T9033), Tu (cat#654380), and β-mercaptoethanol (cat#M6250) from Sigma-Aldrich; and Hoechst 33258 (cat#H3569) from Thermo Fisher.

Techniques: Activation Assay, In Vivo, Injection, Western Blot, Isolation, Activity Assay, Two Tailed Test, Control

Journal: Nature Communications

Article Title: Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

doi: 10.1038/s41467-017-01609-x

Figure Lengend Snippet: Neurotoxin engages ER-p38 MAPK–CMA in mouse brain in a model of PD. a 6-OHDA treatment engages ER-p38 MAPK–CMA pathway in mouse brain. Mice were administrated with 6-OHDA (3 μg) with or without co-injection of SB203580 (2 μg) by unilateral stereotaxic injection to the SN region and killed after 2 days ( n = 16). Pooled SN tissues were analyzed for Bip, p-p38α MAPK, and p38α MAPK with the tissue lysate, and for LAMP2A and p-LAMP2A with the purified lysosomes. b Inhibition of p38 MAPK activity exacerbates 6-OHDA-induced loss of TH (tyrosine hydroxylase) positive neurons (green) in mouse SNc. Mice were stereotaxically injected with saline, 6-OHDA, 6-OHDA plus SB203580, and SB203580 at dosages as described in a . Mouse brain SNc slices were immunostained for TH (scale bar = 200 µm). Right panel shows the quantification of TH positive neurons in each group. c Inhibition of p38 MAPK exacerbates 6-OHDA-induced activation of caspase 3. The SNc slices from mice treated as described in b were immunostained for TH (green) and activated caspase 3 (red) (scale bar = 25 µm). Middle panel shows the quantification, and right panel shows the level of CHOP and cleaved caspase 3 by western blot. Quantifications are shown for a , b , and c ( n = 6 per group. All values are mean ± s.d. (ANOVA with Turkey test. ** p < 0.005 vs. control and ## p < 0.005 vs. 6-OHDA)

Article Snippet: The chemicals and their sources are as follows: SB203580 (cat#5633) from Cell Signaling; 4μ8C (IRE1 inhibitor III, cat#412512), cycloheximide (cat#239763), geldanamycin (cat#345805), and GSK2606414 (PERK inhibitor I, cat#516535) from EMD Millipore; 32 P-ATP (cat#50-905-0808) from Perkin Elmer; 3-methyladenine (3MA, cat#M9281), 6-aminonicotinamide (6-AN, cat#A68203), 6-hydroxydopamine (6-OHDA, cat#H4381), ammonium chloride (NH 4 Cl, Cat#254134), ATP (cat#A2383), BFA (#B7651), DAPI (cat#D9542), MG132 (cat#M7449), Tg (cat#T9033), Tu (cat#654380), and β-mercaptoethanol (cat#M6250) from Sigma-Aldrich; and Hoechst 33258 (cat#H3569) from Thermo Fisher.

Techniques: Injection, Purification, Inhibition, Activity Assay, Saline, Activation Assay, Western Blot, Control