Fig 1.
ULK1 phosphorylates p62 at S409.
A.-C. In vitro p62 phosphorylation assay by ULK1 with purified MBP-p62 WT or mutant proteins. Bacterially expressed MBP-p62 was purified and then MBP tag was cleaved by Factor Xa. The purified p62 was incubated with Myc-ULK1 WT or KI mutant IPed from transfected HEK293T cells at 37°C for 30 min. Phosphorylation of p62 was examined by 32P-labeling and autoradiography or p-S409 specific antibody. A. p62 is an ULK1 substrate in vitro. Alkaline phosphatase(AP) was used to dephosphorylate p62. 32P-autoradiograph shows autophosphorylation of ULK1 and p62 phosphorylation. B. ULK1 phosphorylates p62 at Ser409 in vitro. Purified MBP-p62 WT or S409A proteins were used in ULK1 kinase assay in the presence of 32P-ATP. C. ULK1 phosphorylates Ser409 of p62. Purified MBP-62 WT proteins were incubated with Myc-ULK1 variants isolated from transfected HEK 293T cells. Immunoblotting assay with indicated antibodies, including phospho-p62 antibody against Ser409, was followed. Afterwards, the membrane probed with p-p62 antibody(S409) was incubated with alkaline phosphatase(AP) to dephosphorylate p62. Asterisks indicate nonspecific bands. D. p62 S409 is a ULK1 substrate. HEK 293T cells were transfected wit empty vector, FLAG-p62 WT or FLAG-p62 S409A together with Myc-ULK1 WT or KI. IP with anti-FLAG antibody was performed, followed by Westernblot assay with indicated antibodies.
Fig 2.
Phosphorylation of p62 at S409 is enhanced upon proteasome inhibition.
A. ULK1 mediates p-S409 of p62 upon MG132 treatment. HEK 293T cells transfected with empty vector, Myc-ULK1 WT or KI were treated with MG132 and then cell lysates were analysed with indicated antibodies(Top). The ratio of p-p62 and p62 was obtained by dividing the level of p-p62 by total p62(n = 3). One sample t-test was used and data are represented as mean ± SEM(n = 3). * p < 0.05; ns, not significant(bottom). B. ULK1 is important for MG132-induced p-S409 of p62. WT and ULK1 KO MEFs were treated with MG132 and analyzed with indicated antibodies(Top). The ratio of p-p62 and p62 was obtained by dividing the level of p-p62 by total p62(n = 3). One sample t-test was used and data are represented as mean ± SEM(n = 3). * p < 0.05; ns, not significant(bottom). C. MG132 treatment but not starvation increases p-S409 levels of p62 or ULK1-p62 interaction. p62 KO MEFs stably expressing empty vector or FLAG-p62 were incubated with MG132 or starved for glucose(-glu) or amino acid(-aa). F.M. indicates full medium as a control. Subsequently, IP with anti-FLAG or-ULK1 antibodies were performed and immunoprecipitants were analyzed with indicated antibodies.
Fig 3.
Phosphorylation of p62 at S409 is enhanced upon expression of polyQ-Htt proteins.
A. Expanded polyQ-mCFP induces p-S405 and p-S409 of p62 in a polyQ length dependent manner. HeLa/25Q, 65Q, 103Q—mCFP cells were cultured with or without doxycycline to regulate the expression of each protein. Cellular lysates were assayed with indicated antibodies. GFP antibody was used to monitor induction of polyQ-mCFP proteins. B. The ratio for Fig. 3A was obtained by dividing the level of p-p62 by total p62(n = 3). One sample t-test and student t-test were used and data are represented as mean ± SEM(n = 3). * p < 0.05; ** p < 0.01; ns, not significant C. Shutoff of the expression of 103Q-mCFP reduces p-S405 and p-S409 of p62. Induced HeLa/103Q-mCFP cells were replenished with Doxycycline to turn off the protein expression and then were analyzed with indicated antibodies. D. p62 phosphorylation is increased in z_Q175 HD mouse model. Striatal and cortical lysates of 1, 5, 10, and 15 months old WT and Q175 mice were prepared and analyzed with indicated antibodies(n = 4). E. P-S409 localizes to and surrounds 103Q-mCFP positive inclusions. Induced HeLa/25Q-mCFP and HeLa/103Q-mCFP cells were stained with anti-p62 and p-p62(S409) and examined under confocal microscope. Scale bar = 10 μm.
Fig 4.
Accumulation of protein aggregates induces the interaction between ULK1 and p62.
A. The accumulation of ubiquitinated proteins induces ULK1-p62 interaction. WT and ULK1 KO MEFs lysates treated with or without MG132 were subjected to IP with anti-ULK1 antibody and immunoblotted with anti-ULK1 and-p62 antibodies. Asterisks indicate non-specific bands. B. ULK1-p62 interaction in a p62 stable cell line. p62 KO MEFs stably expressing FLAG-p62 or empty vector were incubated with or without MG132. IP was performed with anti-ULK1 antibody, followed by immunoblot assay using indicated antibodies. C. Co-localization of p62 and ULK1 upon MG132. p62 KO MEFs stably expressing FLAG-p62 WT were treated with MG132, fixed, stained with ULK1(green) and p62(red) antibodies, and visualized under fluorescent microscope. Line profile was used to illustrate co-localization between p62 and ULK1. Green and red lines indicate ULK1 and p62 staining profiles, respectively. Scale bar = 10 μm and 2.5 μm for zoomed images. D. ULK1-p62 interaction increases upon induction of expanded polyQ proteins. HeLa/25Q, 65Q, 103Q—mCFP cells were transfected with empty vector or FLAG-p62 and were cultured with or without doxycycline to regulate the expression of each protein. Cellular lysates were used to perform IP with anti-ULK1 antibody and immunoblot assay with indicated antibodies was followed. GFP antibody was used to monitor induction of polyQ-mCFP proteins.
Fig 5.
ULK1-mediated phosphorylation of p62 at S409 enhances p62 and Ub binding affinity.
A. P-S409 enhances binding between p62 and poly-Ub proteins. Cellular lysates of p62 KO MEFs stably expressing empty vector, FLAG-p62 WT, S409A, or S409E were incubated with p62 KO MEFs lysates treated with MG132 and subsequently subjected to IP using anti-FLAG antibody. Immunoblot assay with indicated antibodies was followed. B. Quantification of the results from Fig. 5A were obtained by normalizing levels of IPed Ub to FLAG blots; then S409A or S409E were normalized to WT. One sample t-test was used and data are represented as mean ± SEM(n = 3). * p < 0.05; ns, not significant C. p62 UBA S409E has an enhanced binding affinity to mono-Ub. Binding affinities of p62 UBA WT(left) or S409E(right) to mono-Ub were measured by Isothermal Titration Calorimetry(ITC). Representative ITC profiles are shown.
Fig 6.
Phosphorylation of S409 destabilizes the UBA dimer interface and regulates phosphorylation of S405.
A. S409E does not influence overall folding of UBA. Overlay of 1H-15N HSQC spectra of 15N-labeled p62 UBA WT(red) and S409E(blue) in the absence of mono-ubiquitin(Ub). B. p62 UBA S409E destabilizes p62 UBA dimer. Differential Scanning Calorimetry was performed with p62 UBA WT and S409E to measure the melting temperature of p62 UBA dimer. C. Overlay of 1H-15N HSQC spectra of 15N-labeled p62 UBA WT(blue) and S409E(red) in the presence of 6-fold excess unlabeled mono-Ub. D. ULK1 phosphorylates p62 at S405 and p-Ser409 is a prerequisite for ULK1-mediated p-S405. HEK 293T cells were transfected with indicated plasmids. IP with anti-FLAG antibody was performed and Westernblot assay with indicated antibodies was followed.
Fig 7.
Phosphorylation of p62 at S409 is required for autophagic degradation of polyubiquitinated proteins and the recruitment of autophagy proteins.
A.-C. p62 p-S409 regulates autophagic degradation of ubiquitinated proteins. p62 KO MEFs stably expressing indicated constructs were treated with MG132 for 16 hr(pre), then media were switched to serum starvation(S.S.), in combination with CQ for 24 hr(post). A. p62 S409A overexpressing cells are resistant to autophagic degradation of poly-Ub proteins. Total cellular lysates were subjected to immunoblot assay with indicated antibodies. B. The level of poly-Ub proteins from Fig. 7A was normalized to actin level, and further to each own control. Student’s t-test was used and data are represented as mean ± SEM(n = 4). * p < 0.05; ns, not significant C. p62 S409A is impaired in the recruitment of autophagy machinery proteins. MEFs were fixed, stained with antibodies against p62(red) and WIPI2, LC3, Rab7, or LAMP2(green), and then visualized under fluorescent microscope. Line profile was used to illustrate co-localization. Green lines indicate WIPI2, LC3, Rab7, or LAMP2 and red lines indicate p62. Scale bar = 10 μm, 5 μm for enlarged images.
Fig 8.
Phosphorylation of p62 at S409 enhances the autophagic degradation of polyQ-Htt mutant proteins.
HeLa/65Q–mCFP cells were transfected with mCherry-p62 WT, S409A or S409E and treated with Rapamycin to induce autophagy. A. Cells were fixed and visualized under fluorescent confocal microscope. Scale bar = 20 μm. B. Quantifications of the results in Fig. 8A were performed by counting cell numbers containing 65Q-mCFP aggregates. Then the number of cell carrying 65Q-mCFP aggregates was normalized to the number of control cells transfected with p62 WT(left panel). Efficiency of polyQ clearance(right panel) was obtained from the ratio of control sample vs. rapamycin-treated sample. C. Cells were separated into detergent soluble and insoluble fractions and probed with indicated antibodies. D. Quantifications were performed by normalizing the intensity to that of control samples transfected with p62 WT(upper panel). Efficiency of polyQ reduction(lower panel) based on the results in Fig. 8C was obtained as described in Fig. 8B. One sample t-test and student t-test were used and data are represented as mean ± SEM(n = 4). * p < 0.05, ** p < 0.01; ns, not significant.
Fig 9.
The working model for ULK1-mediated p-S409 and p-S405 of p62 in selective degradation of ubiquitinated proteins and polyglutamine-expanded proteins.
Accumulated polyubiquitinated(poly-Ub) proteins or polyQ-expanded proteins trigger interaction of p62 with ULK1. This interaction induces ULK1-mediated p62 phosphorylation at S409 in UBA domain, which facilitates dimer to monomer transition of UBA domain, and subsequent phosphorylation at S405(mediated by either ULK1, CK2 or TBK-1). As a result, the phosphorylation of p62 at S405 and S409 leads to enhanced binding affinity of p62 to poly-Ub or polyQ-expanded proteins. The presence of ULK1 and p62 p-S405 and p-S409 in poly-Ub or polyQ-expanded protein aggregates causes the recruitment of autophagy machinery that is responsible for the degradation of poly-Ub or polyQ-Htt mutant proteins.