CCDC152: A Golgi Protein That Regulates The Cell G1/S Restriction Point

Introduction:CCDC152(Coiled-coil Domain Containing Protein 152)belongs to the CCDC family.It contains four coiled-coil domains which are required for protein-protein interactions,but its function is unknown.In eukaryote cells,some Golgi proteins which contain the Coiled-coil domains are referred as "Golgins".Golgins were originally identified as a group of proteins in the sera from patients with various autoimmune diseases,and they are named in different Golgins according to their different molecular weight.Golgins and GRASP are considered to form a structure of Golgi(or Golgi matrix)and play important roles in the structure maintenance and function of Golgi apparatus..Recent studies found that the Golgins can regulate the cell cycle by regulating of cell cycle checkpoint related kinases,therefore Golgi appratus is also referred as "Golgi checkpoint".This is a new mechanism that cell cycle is regulated by the integrity of Golgi apparatus but not DNA damage activated cell cycle blockage.Although the mechanism of the regulation of Golgi fragmentation in cell cycle is not yet clear,these findings sufficiently explain the inheritance of organelles and signaling pathways in the regulation of cell division.Checkpoint kinase 1(Chkl)/CDC2 and pRb(retinoblastoma protein)are critical kinases of cell cycle checkpoint.CDC2 is a serine/threonine protein kinase identified in fission yeast and pRb is encoded by a tumor suppressor gene Rb.E2F,a transcription factor,can bind to the targeted DNA and activate transcription.pRb and E2F can form a pRb-E2F complex and thus sequester the effect of E2F.Studies have shown that CDC2 can phosphorylate pRb at Ser807/811 residues to inactivate pRb and release the E2F.Therefore,CDC2-pRB-E2F forms a positive feedback loop.When CDC2 protein is phosphorylated at residues T14/Y15,the kinase activity is inhibited and thus resulting in an increased pRb-E2F complexes,DNA transcription inhibition and cell cycle arrest.Our current research implies that CCDC 152 is a Golgi protein and has a role in regulating of G1/S phase transition in cell cycle.Materials and Methods:(1)To study CCDC 152 protein subcellular localization:Firstly,the 293T cells were grown on coverslips in 24-well plates and fixed,then analysised the position and morphology of endogenous CCDC152 in the cell cycle by immunohistochemistry;Secondly,CCDC152 yellow fluorescent expression vector(CCDC152-YFP)had been constructed,293T cells were transfected with CCDC152-YFP to observe exogenous CCDC152 expression in the cell cycle.Then compared if the endogenous CCDC152 expression was consistent with exogenous;Thirdly,293T cells were transfected with Golgi marker vector of expressing yellow fluorescent(Golgi-YFP)and CCDC152-YFP,after 48 h,the cells were treatment with colchicine or BFA to observe the changes of CCDC152 and Golgi expression;Further to observe the changes of Golgi marker expression after interfering CCDC152 expression.(2)To study cell cycle regulation by CCDC152:Firstly,293 cells were transfected with CCDC152 overexpression vecter or Si-CCDC152,48h post-transfection,cell cycle analysis was performed by flow cytometry methods.Secondly,293T cells were grown on coverslips in 24-well plates,then transfected 293T cells with CCDC152 overexpression vector or si-CCDC152 to observe the BrdU incoperation by immunohistochemistry.(3)To study the mechanisms of cell cycle regulation by CCDC152:293T cells were transfected with CCDC152 overexpression vecter and si-CCDC152,after 48 h,to assess pRb-S807/811 and CDC2-Y15 kinase expression by Western Blot.Results:(1)CCDC152 orchestrates with cell cycle progression.The expression of endogenous CCDC152 changes in the cell cycle.During the interphase,CCDC152 appeared as a large continuous perinuclear formation;When cells in mitosis,CCDC152 dispersed throughout the cytoplasm.Further found that exogenous CCDC152 expression changes were consistent with endogenous CCDC152.Effect of Brefeldin A(BFA)and Colchicine on pEYFP-Golgi and pEYFP-CCDC152 changes.We found that both drugs can cause CCDC152 disperse throughout the cytoplasm.Golgi marker expression changes were Consistent with CCDC152.Interferencing CCDC152 to observe the changes of Golgi apparatus found that pEYFP-Golgi dispersed throughout the cytoplasm.(2)Overexpression CCDC152 induced the cell number of G1 phase decreased and S phase increased;Si-CCDC152 maked G1 and S phase of the cell cycle change not obviously.BrdU incoperation experiment showed that overexpression of CCDC152 induced DNA synthesis;while interferring CCDC152,DNA synthesis is enhanced more obviously.These results suggest that CCDC152 has a role in regulating cell G1/S phase transition.(3)Overexpsion of CCDC152,the kinase pRb phosphorylation at residues S807/811 was up-regulated,whereas interference CCDC152,the phosphorylations were also up-regulated;while the overexpression or interference CCDC152,the phosporylation of CDC2 kinase at residue Y15 was down-regulated.Whereas the expression of p53 does not change very obvious after interferencing or overexpression of CCDC152.This shows that CCDC152 regulates G1/S phase transition by activating the phosphorylation of pRb,CDC kinase.Conclusions:(1)CCDC152 is a Golgi protein.(2)CCDC152 regulates cell cycle by phosphorylation of pRb and CDC kinase.(3)CCDC152 regulates G1/S transition.Therefore,our study provides a new evidence that Golgi complex could regulate the cell cycle.