The Dephosphorylation And Biological Functions Of C-Myc By Phosphatase SCP1and SCP1Membrane Localization And Palmitoylation

c-Myc is a highly conserved, B/HLH/Zip domain containing oncoprotein. It can regulate its downstream genes transcription through forming a heterologous dimer. c-Myc has multiple functions in human cells, such as regulate cell cycle, proliferation, metabolism, apoptosis, transforming, genomic instability and angiogenesis. In addition, c-Myc also plays an important role in cell differentiation, stem cell self-renew, adipocyte differentiation and tumor genesis.SCP1is Carboxy-terminal domain RNA polymerase Ⅱ polypeptide a small phosphatase1. It is also called NLI-IF, SCP1, and the phosphatase very highly conserved from flies to mammals that can recognize specific SP phosphorylation site in its target. It has been reported that, SCP1can repress neuronal gene expression in non-neuronal cells. And it can also perform transcript suppression by regulate RNA polymerase activity. Meanwhile, SCP1can also promote cell migration by stable Snail protein. Although recently, some studies have shown that SCP1has a tumor suppressior function. The detailed mechanism and its new biological function have not been revealed.In this paper, we used technics including bioinformatics, cell biology and molecular biology to screen the new phosphatase regulates c-Myc S62dephosphorylation. We find that SCP1can dramatically dephosphorylate c-Myc S62and promote its protein instability. At the same time, we find that besides its nucleus localization, SCP1also localizates in the cell membrane, and the molecular mechanism is also discussed.The chapters in part one:I. SCP1regulates c-Myc Ser62dephosphorylationWe took advantage of a phosphatase library to screen a potential phosphatase that can dephosphorylate Ser62of c-Myc, and used a Ser62phosphorylation antibody to test the phosphorylation status. Then we screened that SCP1can notably dephosphorylates the c-Myc. Then we conformed the result by a series of in vivo and in vitro dephosphorylation assays. Ⅱ. the HLH domain of c-Myc can bind the c-tail of SCP1We used in vivo and in vitro assays to show that overexpressed c-Myc and SCP1can be interation each other, as is the endogenous proteins. Through the construct of SCP1deletion mutant, we find out that c-Myc can interact with the c-tail of SCP1. Meanwhile, the deletion mutant of c-Myc showed that the c-term of c-Myc is also important for its interaction with SCP1.Ⅲ. SCP1regulates c-Myc instability by dephosphorylationIt has been reported that phosphorylation of Ser62is important for its protein stability. Both overexpression and knockdown assay showed that, SCP1can promter c-Myc protein instability through specifically dephosphorylation of c-Myc Ser62. In addition to, we discovered that C-tail of SCP1is of great importance to its phosphatase activity. And Tyr251and Ser252is the critical site.Ⅳ SCP1suppress liver tumor cell proliferation by promote c-Myc Ser62dephosphorylation We tested SCP1protein expression in mouse organs, and it’s highly expresstion in liver. Then we chose normal mouse liver cell and human liver tumor cell and find significant negative correlation between SCP1and c-Myc. What is more, as the expression of SCP1decreases, liver tumor cells proliferation increases.The chapters in part two:Ⅰ. the cell membrane localization of SCP1Unexpected that, we find SCP1localizates on the cell membrane through immunofluorescence assay. And we tested endogenous SCP1and overexpressed SCP1through cell fraction assay, and confirm our findings. The following cell fraction and immunofluorescence of the deletion mutant of SCP1showed that the N-terminal31-55animo acid is critical for its cell membrane localization.Ⅱ. Palmitoylation of SCP1Bioinformatics analysis suggested that C44, C45and C47are the potential palmitoylation sites which cause SCP1membrane localization. First, we use point mutation of SCP1and palmitoylation inhibitor to indirectly show the possibility that SCPl can be palmitoylated. Then, to test this hypothesis we used to directly prove palmitoylation by ABE assay and3H palmitoylation assay.In a word, after we screen the phosphatase library and use the approaches of Cell Biology, Biochemistry and Molecular Biology, we successfully identified SCP1as a new phosphatase that regulate dephosphorylation of c-Myc. We first discover that tumor suppressor SCP1works as a negative regulator of c-Myc protein, which provide new clues and ideas for further understanding the protein instability and biology function of c-Myc. We mainly focused on the regulation of the c-Myc protein instability by SCP1.SCP1is discovered, as a tumor suppressor, dephosphorylate c-Myc through a phosphorylation-dependent pathway. The transcription activity of c-Myc is suppressed on protein levels by SCP1, which restrain the proliferation of liver cancer on cell levels. c-Myc plays an important role in tumorgenesis. It’s the significance of discovering SCP1as a negative regulator of c-Myc on protein level that provide a new target for the treatment of cancer and other diseases. Furthermore, the discovery of SCPllocaliaztes in the cell membrane through palmitoylation, that is the first reported investigation about the relationship between serine and threonine phosphatase and membrane localization, which indicates that SCP1influence cellular regulation through more mechanism.