Identify And Functional Study On Two Isoforms Of Human Smurf1

Objective:Identify and functional study on two isoforms of human Smurfl.Method:Analyze protein expression level of two isoforms Smurfl (Smurfl L and Smurfl S) in mouse tissues and9cell lines. The mRNA level of the two isoforms were edtected by RT-PCR in12human adult tissues cDNA libraries and9human fetal tissues cDNA libraries. Using indirect immunofluorescence assay (IIF) to analyze the subcellular localization of Smurfl L/S protein. Smad5was transfected into HEK293T cells with an increased amount of Smurfl L/S, followed by Western blotting to detect the degradation of Smad5. Co-immunoprecipitated Smad5/CKIP-1with Smurfl S and Smurfl L to detect the change of interaction. In vitro ubiquitination assay was used to analyze the ubiquitination modufication of Smurfl L/S.Result:1.Mouse tissue expression profile analysis showed that the Smurfl S was mainly expressed in heart, liver, spleen, lung, kidney, stomach, small intestine, brain, uterus, bladder, pancreas and skeletal muscle, and Smurfl L mostly expression in heart, liver, lung, stomach and skeletal muscle. Cell lines expression profile analysis showed that Smurfl S were highly expressed in Cell lines of HCT116+/+, HCT116-/-, MCF-7, Hela, HEPG2, Chang, HEK293T, U2Os and HI299, but Smurfl L mainly expression in Cell lines of HCT116+/+, Chang, U2Os and H1299. Isolate Smurfl from12human adult tissues cDNA libraries and9human fetal tissues cDNA libraries, and the results showed Smurfl S exist in almost all of tissues, while Smrufl L expressed in lung, kidney, stomach, ovary, skeletal muscle, spinal cord and mammary gland of human adult tissues and heart, stomach, lung, spleen and mammary gland in human fetal tissues.2. Most of Smurfl S exist at the cytoplasm, but Smurfl L mainly at the membrane.3. Protein levels Smad5decreased with an increased amount of SmurflS. However, at the exist of SmurflL, the expression of Smad5increased at first and then decreased sharply.4. Smurfl S was stronger than Smurfl L in the physical interaction withSmad5/CKIP-l.5. The ubiquitination modification of SmurflS was stronger than Smurfl L.Conclusion:1、The distribution and expression of Smurfl S and Smurfl L were different. Smurfl S was expressed in all of tissues, and Smurfl L was specific expressed in heart, lung, stomach and skeletal muscle.2、It is different of the subcellular localization of Smurfl S and Smurfl L3、Substrate hydrolysis of Smurfl S and Smurfl L were Significant differences.4、The26residues of Smurfl L affect interactions between Smurfl and its substrates by binding to WW domain.