Effect And The Related Mechanisms Of Phosphorylation On UBC12

Introduction:Neddylation is a process of conjugating the ubiquitin-like molecule NEDD8 to the targeted proteins.This process is very similar to ubiquitination,catalyzing by NEDD8-activationg enzyme E1(NAE),NEDD8-conjugating enzyme E2s(UBC12 or UBE2F)and substrate-specific NEDD8-E3 ligases.Neddylation pathway plays an important role in many aspects in cells,such as cell cycle,cell proliferation,cell metabolism,and cell differentiation.UBC12 is the first characterized E2 enzyme in neddylation pathway and mediates neddylation of most reported substrates including Cul-1-4.As E2s accept NEDD8 from E1 and coordinate NEDD8 with downstream steps in the cascade,E2s play a central role in neddylation pathway.Despite the fact that UBC12 is an E2 enzyme in a post-translational modification pathway,UBC12 itself is regulated by post-translational modification at the same time.Phosphorylation is the most common post-translational modification,which plays a vital role in regulating and finely tuning a multitude of biological pathways.Therefore we wonderer wether phosphorylation of UBC12 would have important influence on function of UBC12.Objective:In this study,we are trying to figure out whether UBC12 could be phosphorylated,discover the mechanisms that regulate UBC12 phosphorylation,and find out how phosphorylation of UBC12 regulates its function.We hope to enrich post-translational modifications of UBC12,and provide new theory and new method for design of neddylation inhibitors which targets UBC12.Contents and Methods:Our research was mainly divided into three parts:(1)Investigate whether UBC12 could be phosphorylated and find the phosphorylation sites on UBC12:Immunoprecipitation and immunoblotting were used to detect phosphorylation of UBC12;Phos-tag immunoblotting was used to detect phosphorylation of UBC12;Mass spectrum was used to detect phosphorylation sites of UBC12;Point mutation technology and phos-tag immunoblotting was used to search for phosphorylation sites of UBC12;Point mutation technology,immunoprecipitation and immunoblotting were used to confirm phosphorylation sites of UBC12.(2)Reveal the kinase and phosphatase of UBC12 and figure out the regulatory mechanism of UBC12 phosphorylation:Kinase activators and kinase inhibitors were used to search for kinase of UBC12 phosphoryaltion;Immunoprecipitation and immunoblotting were used to detect whether overexpressiono of PKCa CAT,PKCβⅠCAT and PKCβⅡCAT could promote phosphorylation of UBC12;Immunoprecipitation and immunoblotting were used to detect whether PKCα could promote phosphorylation of UBC12;In vitro PKC assay was used to detect whether PKCα could phosphorylate UBC12;shRNA was used to knock down subtypes of isoforms constituting PP1 or PP2A,followed by immunoprecipitation and immunoblotting that were used to detect whether PP1 or PP2A was the phosphatase of UBC12.(3)Investigate the effect of UBC12 phosphorylation on neddylation and reveal the related mechanism:Immunoprecipitation and immunoblotting were used to detect the interaction between UBC12 and E1 enzyme UBE1C or E3 enzyme DCN1;Immunoprecipitation and immunoblotting were used to detect influence on interaction between UBC12 and UBE1C under treatment of TPA and/or calyculin A;In vitro NEDD8 transfer assay was used to detect the NEDD8 transfer ability of UBC12 WT/S6A/S6D/S6E.Results:1.Phosphorylation and phosphorylation sites of UBC12:Firstly,we wanted to find out whether UBC12 could be phosphorylated:Our data showed serine/threonine sites of UBC12 could be phosphorylated;We then searched for phosphoryation sites of UBC12:Phosphorylation sites detected by both mass spectrum experiments are S6,S28 and S175;Phos-tag assay was used to search for phosphorylation sites of UBC12 and S6 was found to be the possible phosphorylation site of UBC12;Immunoprecipitation also showed S6 could be the phosphorylation site of UBC12.2.Regulatory mechanisms of UBC12 phosphorylation:We have proved UBC12 S6 could be phosphorylated in the first part of research.In this part,we are going to reveal the regulatory mechanism of UBC12 phosphorylation,including finding the kinase and phosphate of UBC12.We firstly tried to find out the kinase of UBC12:Our reaults demonstrated that PKC activator TPA could promote phosphorylation of UBC12;PKC inhibitors could inhibit phosphorylation of UBC12;overexpression of PKCα-CAT could obviously increase phosphorylation of UBC12;In vitro PKCa kinase assay showed UBC12 WT could be phosphorylated by PKCa in vitro.These data indicated that PKCa was the main kinase of UBC12.We then tried to find out the phosphatase of UBC12.Our data showed phosphorylation of UBC12 was increased when PPP2R1A(subunit of PP2A)was knockdown by lentivirus-shPPP2R1 A,indicating that PP2A was the phosphatase of UBC12;lentivirus-shPPP1CA,shPPP1CB,and shPPP1CC(three isoforms of the catalytic subunit of PP1)did not increase the phosphorylation of UBC12,indicating PP1 was not the phosphatase of UBC12.3.Influence of UBC12 phosphorylation and related mechanisms:In the first two parts of research,we have found UBC12 S6 could be phosphorylated and revealed the kinase and phosphatase of UBC12.In this part of research,we continued to discover the effect of UBC12 phosphorylation and related regulatory mechanisms.Our experiments showed UBC12-HA WT/S6A could interact with UBE1C but UBC12 S6D/S6E showed an obviously weaker interaction with both UBE1C,compared to UBC12 WT/S6A;PKC activator TP A inhibited interaction between UBC12 and UBE1C modestly;Calyculin A inhibited interaction between UBC12 and UBE1C.These results indicated phosphorylation of UBC12 inhibited interaction between UBC12 and E1 enzyme UBE1C.We further investigated whther phosphorylation of UBC12 could influence NEDD8 transfer from UBE1C to UBC12.Our results showed UBC12 S6D and UBC12 S6E,which simulated hyperphosphorylated UBC12,could only receive much fewer NEDD8 in same period of time compared with UBC12 WT and UBC12 S6A;UBC12 S6D and UBC12 S6E demonstrated much lower NEDD8 transfer ability when compared to UBC12 WT/S6A at the same concentration.These results indicated that phosphorylation of UBC12 inhibit its ability to receive NEDD8 from E1 enzyme:Conclusion:Our research found UBC12 Ser-6 could be phosphorylated,revealed the regulation mechanism of UBC12 phosphorylation,and demonstrated that phosphorylation of X UBC12 could inhibit NEDD8 transfer through inhibiting interaction between UBC12 and UBE1C.Our research enriched post-translational modifications of UBC12 and provide information to find new strategy of intervening neddylation pathway.