The Cell Cycle Dependent Translocation Phenomenon Of CacyBP/SIP And Its Regulation Mechanism

The calcyclin binding protein was firstly found as a target of S100A6protein byFilipek et al in Ehrlich ascites tumor cells of mice and later was round in human cells andlater on was found by another group as Siah-1interacting protein which was abbreviatedSIP in human cells. There is93%homology between CacyBP and SIP after sequenceanalysis, which suggested that SIP is equivalent to CacyBP. CacyBP/SIP contains229amino acids and the molecular weight of it is about30KD. The previous research hadfound that CacyBP/SIP could bind with some proteins of S100family as calciumdependent. CacyBP/SIP is highly expressed in nervous tissues, which indicated thatCacyBP/SIP is related to neural differentiation. CacyBP/SIP also could bind with Siah-1and Skp1, which together into an ubiquitin ligase complex and take part in the degradationof β-catenin and then participate in the development of malignant tumors. A latest studyfound that CacyBP/SIP closely correlated with the development and progression of alzheimer disease. The study alaos found that CacyBP/SIP is mainly located in cytoplasm,but translocate to nuclear membranes and nucleus after the elevation of Ca2+or activationof PKC. Our group had found that the overexpression of CacyBP/SIP could inhibit theproliferation of renal cancer cells and gastric cancer cells, gene chip showed that,CacyBP/SIP could down-regulate the expression level of cyclin B3and cyclin A1, so weconjecture that CacyBP/SIP is a cell cycle regulate molecule which affect the cell divisioncycle especially G2/M phase by degrade proteins or other ways. Because the subcellularlocalization of protein molecule is close relate with its function. This work aims to obversethe subcellular locatization, protein expression and phosphorylation levels of CacyBP/SIPin different cell cycle phase, and then obverse the location and expression change of theother proteins of CacyBP/SIP ubiquitin ligase complex, which reveal the possible role ofCacyBP/SIP in regulation of cell cycle and lay a good foundation for elucidating theantineoplastic mechanism of CacyBP/SIP.【Purpose】1. Observe the subcellular locatization and expression levels ofCacyBP/SIP protein in different phases of gastric cancer cells SGC7901.2. Study thephosphorylation level of CacyBP/SIP in different cell cycle phases.3. Observe thesubcellular locatization and expression levels of ubiquitin ligase complex members Siah-1,Skp1and the S100A6protein of CacyBP/SIP ligand in different phases of gastric cancercells SGC7901, then study the interaction ability of Siah-1and Skp1with CacyBP/SIP andthe expression change of β-catenin.【Methods】1. Synchronize the cell cycle of gastric cancer cells SGC7901by themethod of double thymidine blocks and nocodazole blocks, confirm the effect of blocksby flow cytometry. Observe the subcellular locatization of CacyBP/SIP in different cellcycle phases by the method of immunofluorescence staining. Detect the expression levelof CacyBP/SIP protein by western blot.2. Detect the phosphorylation status ofCacyBP/SIP in different phases of cell cycle and its function by the methods ofimmunoprecipitation and western blot.3. Observe the subcellular locatization of ubiquitinligase complex members Siah-1, Skp1and the S100A6protein of CacyBP/SIP ligand indifferent cell cycle phases by immunofluorescence staining. Detect the expression level of them by western blot. Study the interaction ability of Siah-1and Skp1with CacyBP/SIPby immunoprecipitation and western blot. Study the expression change of β-catenin bywestern blot.【Result】1. The result of flow cytometry showed that most of SGC7901cells wereblocked in G1/S phase by double thymidine blocks, withdrawal and culture4h and thenthe cells are in S phase. Most of cells get into G2phase in8to12h and re-enter G1phasein16h. Also it is showed that most of cells were blocks in G2phase by nocodazole blocks,the cells mainly distribute in G2in0h after withdrawal and culture, most of cells get intoG1/S phase in2h,4h and6h. Immunofluorescence staining found that CacyBP/SIP ismainly distribute in the cytoplasm in G1/S phase, but gather into perinuclear or nuclear inG2phase. Western blot analysis showed that the expression of CacyBP/SIP have noobvious change in each pahse of cell cycle in total protein level. In nuclear protein level,the expression of CacyBP/SIP in G2phase is higher than G1/S phases.2.Immunoprecipitation and western blot showed that the phosphorylation level of nuclearCacyBP/SIP is elevated when it translocate to nucleus.3. To study the possible cell cycleregulation mechanism of CacyBP/SIP, we first detected if the CacyBP/SIP ubiquitin ligasecomplex members Siah-1, Skp1exist the location change of cell cycle related.Immunofluorescence staining showed that the ubiquitin ligase complex members Siah-1,Skp1is mainly distribute in the cytoplasm in G1/S phase, but gather into nuclear in G2phase, which is in line with CacyBP/SIP localization. Western blot analysis showed thatthe expression of Siah-1, Skp1have no obvious change in each phase of cell cycle in totalprotein level. In nuclear protein level, the expression of Siah-1, Skp1in G2phase is higherthan G1/S phases. We also obverse the interaction ability of Siah-1and Skp1withCacyBP/SIP in nucleus, the result showed that binding ability of each other is higher in G2phase than G1/S phase. Based on previous reports about the ubiquitin ligase complexcould degrade non-phosphorylated β-catenin, we study the expression of β-catenin innucleus in different phases and found that expression of β-catenin is negative correlatewith the expression of ubiquitin ligase complex. As a ligand of CacyBP/SIP, S100A6didn’t have the location and expression change. 【Conclusion】1. CacyBP/SIP have a translocation phenomenon of cell cycledependent, the total protein of CacyBP/SIP have no obviously change, but increased in thenucleoprotein level, which demonstrate that the CacyBP/SIP protein didn’t change theexpression level, but just change the location.2. The phosphorylation of CacyBP/SIP isincreased when it translocate to nucleus, which suggest that the activity of CacyBP/SIPhas changed.3. The CacyBP/SIP ubiquitin ligase complex members Siah-1and Skp1alsohave a translocation phenomenon of cell cycle dependent, the total protein of Siah-1andSkp1have no obviously change, but increased in the nucleoprotein level. These resultsdemonstrate that they didn’t change the expression level, but just change the location,which is in line with CacyBP/SIP. It is showed that CacyBP/SIP, Siah-1and Skp1form anubiquitin ligase complex and change the activity when they translocate to nucleus, but theexpression of β-catenin is negative correlate with ubiquitin ligase complex, which indicatethat β-catenin is degraded by ubiquitin ligase complex in nucleus, at least the part of thedegradation was conducted through this way. As a ligand of CacyBP/SIP, S100A6proteindidn’t have obvious change, which demonstrate that it didn’t participate in the relatefunctions.