Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates (original) (raw)
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.