TI - Kinase -dead ULK mutants inhibit autophagy . AB - In yeast , Atg1 kinase activity is up-regulated during autophagy induced by nitrogen starvation or Tor inactivation . However , it is not known whether this is the case in mammalian cells . We therefore determined ULK kinase activity in MEFs under nutrient-rich and starved conditions . Endogenous ULK1 was precipitated with anti-ULK1 antibody , and the resultant immunoprecipitate was analyzed by an in vitro kinase assay using myelin basic protein as a model SUBstrate . The ULK1 kinase activity level under the starvation condition is increased 1.3-fold relative to the nutrient-rich condition ( Fig 2 A ) . Although this change was modest , these data suggest that the increase in ULK1 kinase activity might be important for the induction of autophagosome formation . We next examined the importance of the ULK kinase activity using ULK kinase -dead mutants . Overexpression of kinase -dead Atg1 mutants inhibits autophagy in D.discoideum and D.melanogaster , whereas overexpression of wild-type Atg1 accelerates autophagy in D.melanogaster . However , in mammalian cells , both wild-type and kinase -dead ULK1 suppress autophagy , as judged by the LC3 conversion assay . We therefore carefully examined the effect of wild-type and kinase -dead mutants of ULK1 and 2 on Atg16L1 puncta formation . In transient transfection experiments , moderate expression of kinase -dead HA-ULK1K46N and HA-ULK2K39T efficiently suppress the starvation-induced Atg16L1 puncta formation ( Fig 2 C ) , whereas wild-type ULK1 and 2 showed almost no effect ( Fig. 2 B ; note that HA-ULK dots are not as clear as those in stable transformants [Fig 1] because of high cytoplasmic signals caused by overexpression ) . In contrast , when overexpressed in higher levels , both wild-type and kinase -dead ULKs suppressed Atg16L1 puncta formation ( unpublished data ) . The shape of wild-type ULK-overexpressing cells was abnormal ( Fig . S3 , available at http : . . . . . . . /jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200712064/DC ) , as previously demonstrated by Chan et al . . The cells generated protrusions and , finally , detached from the culture dish , which is consistent with the previous results in D.melanogaster that Atg1 overexpression caused apoptotic cell death . However , these abnormalities were not observed in cells overexpressing kinase -dead ULK1 and 2 ( Fig S3 ) . Therefore , the effects of overexpression of wild-type and kinase -dead ULKs are different . The kinase -dead mutants indeed act as dominant-negative mutants , whereas wild-type overexpression may cause cytotoxicity by some other mechanism . Collectively , these data suggest that the kinase activity is important for the involvement of ULKs in autophagy . We also generated NIH3T3 cells stably expressing GFP-ULK1K46N and GFP-ULK2K39T . Although both Atg16L1 and GFP-ULKs formed puncta in wild-type NIH3T3 cells after starvation , these puncta were only very rarely observed in NIH3T3 cells stably expressing GFP-ULK1K46N or GFP-ULK2K39T ( Fig 2 D ) , confirming that kinase -dead ULKs function as a dominant-negative mutant in autophagosome formation . We next measured this effect by the LC3 conversion assay . Conversion of cytosolic LC3 ( LC3-I ) to membrane-bound phosphatidylethanolamine ( PE ) -conjugated LC3 ( LC3-II ) occurs during autophagy , and the amount of LC3-II is correlated with the number of autophagosomes . This LC3 conversion during starvation was markedly suppressed in NIH3T3 cells stably expressing ULK1K46N ( Fig 2 E ) , as recently reported by Chan et al . . Another assay was conducted to monitor autophagy flux . Because p62 (SQSTM1/sequestosome 1) can bind LC3 , p62 is preferentially incorporated into autophagosomes and degraded by autophagy . The amount of p62 can therefore serve as a good indicator of autophagic activity . The base level of p62 was up-regulated in ULK1K46N -transfected cells compared with mock-transfected cells . Although the level of p62 decreased during starvation in mock-transfected cells , the decrease was modestly suppressed in ULK1K46N -transfected cells . These data suggest that autophagic flux was attenuated by expression of the kinase -dead ULKs .