TI - FIP200 is required for ULK puncta formation and is important for the stability and proper PHOSphorylation of ULK . AB - Given that FIP200 interacts with ULK1 and 2 , we postulated that ULKs and FIP200 function together . We first examined the membrane targeting of ULKs in wild-type and FIP200-/- MEFs . As we demonstrated in Fig. 1 , GFP-ULK puncta were formed in wild-type MEFs during starvation . These puncta represent the isolation membrane ( Fig 8 A , left ) . However , these dots were never observed in FIP200-/- MEFs , suggesting that puncta formation of ULK1 and 2 depends on FIP200 ( Fig 8 A , right ) . However , this observation may simply reflect impairment of isolation membrane formation in the absence of FIP200 , because even GFP-Atg5 dot formation was suppressed in FIP200-/- MEFs ( Fig 5 C ) . To explore more direct functional connections between ULK and FIP200 , we next examined the expression status of ULK1 in wild-type and FIP200-/- MEFs . The expression level of ULK1 in FIP200-/- MEFs was much lower than in wild-type MEFs under both nutrient rich ( Fig 8 B ) and starvation ( not depicted ) conditions . The ULK1 mRNA expression was enhanced after starvation , but there was no difference between wild-type and FIP200-/- cells ( Fig . S4 A , available at http : . . . . . . . /jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200712064/DC ) . However , we observed faster decay of ULK1 protein in FIP200-/- cells after cycloheximide treatment , suggesting that ULK1 is destabilized in the absence of FIP200 ( Fig S4 B ) . Additionally , we found that ULK1 was detected as a smeared band with faster mobility in FIP200-/- MEFs . As previously reported , we observed that mobility of ULK1K46N is faster than that of wild-type ULK1 , suggesting that ULK1 is autoPHOSphorylated ( Fig 3 D ) . Consistent with this , when we treated ULK1 immunoprecipitated from wild-type cells with lambda phosphatase , ULK1 migrated to a lower position that was suppressed in the presence of phosphatase inhibitors , confirming that ULK1 is PHOSphorylated ( Fig 8 C ) . Likewise , the phosphatase treatment of ULK1 from FIP200-/- cell lysates produced a band with the same mobility ( Fig 8 C , compare lanes 2 and 5 ) , indicating that the faster migration of endogenous ULK1 in FIP200-/- MEFs represents reduced PHOSphorylation rather than other modifications such as protein processing . Therefore , FIP200 is likely important for maintenance of the ULK1 kinase activity . All these results suggest that FIP200 and ULK1 are functionally connected and that this complex plays an important role in autophagosome formation .