Studies On The Neddylation Modification Of HECT-type Ubiquitin Ligase Smurf1

HECT type E3s exert important functions in bone homeostasis, cancer andretroviral budding, however, how its activity is regulated remains unclear. It has beenwell evidented that modification of Cullin-RING ubiquitin ligases by Nedd8promotessubstrate ubiquitination, but the relationship between HECT type E3and Nedd8isunclear. We identified the ubiauitin-like protein Nedd8as an interactor candidate ofHECT type E3Smurf1. Here, we show that Nedd8could enhance the E3ligase activityof Smurf1. Nedd8regulates the function of Smurf1via its neddylation ability. Smurf1iscovalently modified by Nedd8via itself both in vivo and in vitro. In addition, Smurf1promotes neddylation of itself in vitro. The following study found that Smurf1directlybinds Nedd8E2Ubc12in vitro. Deletion analysis indicated that the HECT N-lobeSmall-subunit region of Smurf1could interact with Smurf1directly, which was thesame of Ub-E2UbcH5C. Smurf1showed reduced binding to Ubc12ΔN26andH88-D89A mutant. Our data provide evidence to show that Nedd8conjugation ofSmurf1appeared to depend on the cysteine residue426in the HECT domain. Themutant of C426distinctly affected the ubiquitin E3ligase activity of Smurf1on itssubstrates, such as Smad5,RhoA. Smurf2is also shown to have the similar function likeSmurf1. We demonstrated for the first time that Smurf1is itself modified with Nedd8with very similar characteristics to the autoubiquitination activity of itself, and itsneddylation exerts critical functions in the E3activity regulation.In order to search for new substrates of Smurf1, we performed a yeast two-hybridscreening with the WW domains of Smurf1as the bait and identifiedKLF2(Krüppel-like factor2) as one potential interacting partner. KLF2has beendemonstrated to be essential for normal lung development, T-cell differntiation,migration. Here we show that Smurf1interacts with and targets KLF2forpoly-ubiquitination and proteasomal degradation specifically in lung cancer H1299cells.Smurf1represses the transcriptional factor activity of KLF2and regulates itsdown-stream genes such as CD62L and Wee1.Except for main study around Smurf1, we also demonstrate that ectopic expressionof NuSAP lead to mitotic arrest observably dependent on the kinase activity of ATM.When endogenous ATM was depleted or its kinase activity was inhibited, NuSAP couldnot cause mitotic arrest. We futher show ATM interacts with NuSAP and phosphorylates NuSAP on Serine124. The phosphorylation and interaction occurspecifically at M-phase. Moreover, we investigated the substrate of RBBP6ligase andrevealed the molecular mechanism of RBBP6function in cell cycle regulation. Wefound that RBBP6interacts specifically with the transcriptional repressor ZBTB38, andfunctions as an E3ubiquitin ligase to target ZBTB38for degradation. Depletion ofRBBP6by RNA interference causes the appearance of DNA damage and anaccumulation of cells in G2/M. These effects are rescued by the simultaneous depletionof ZBTB38. Considering how RBBP6deficiency resulted in the embryonic lethalityremains unclear, our research will provide some clue to search for the crital reason.