FIGURE 4.

Stimulation of ULK1 kinase activity by ATG13 and ULK1. A, ATG13 stimulates ULK1 kinase activity. 10 nm recombinant ULK1 was incubated with the indicated amounts of recombinant FLAG-ATG13 in the presence of 0.3 mg/ml MBP and [γ-³²P]ATP as described under “Experimental Procedures.” Reactions were stopped by the addition of SDS sample buffer followed by SDS-PAGE and transfer to nitrocellulose. The upper panel shows the autoradiograph of ³²P-labeled MBP with the total amount of MBP shown by Ponceau-S staining of the membrane below. The quantities of ULK1 and FLAG-ATG13 in each reaction are indicated by immunoblot in the lower panels. B, ATG13 and FIP200 act additively to stimulate ULK1 kinase activity. 10 nm ULK1, 200 nm FLAG-ATG13, or 200 nm hisFIP200 were incubated alone or in combination, as indicated, along with MBP and [γ-³²P]ATP and processed as in A. C, ULK1 kinase activity is impaired in ATG13- and FIP200-depleted cells. MEF cells, either control-, ATG13-, or FIP200-depleted were incubated in complete medium or amino acid-free medium for 1 h, followed by lysis and immunoblotting of FIP200 (top panel) and ULK1 (bottom panel). Protein loading of the duplicate samples is shown by Ponceau-S staining in the lower panel.