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