The Interaction Between Erbin And Plk1Kinase

BackgroundMitosis is an important characteristic of cell life activities, it has been concerned since it wasfound in the late nineteenth century till now. Scientists found that cell mitosis is a complicated and delicateregulation process which was affected by time and space during the lengthy exploration and research. Thecell mitotic abnormalities lead to aneuploidy cells increasing and are bound up with the occurrence anddevelopment of tumor.After a long process of evolution, cells have the checking mechanisms which ensure thesuccessful completion of the cell cycle, DNA replication and chromosomes assignment. The checkingmechanism was called the cell cycle checkpoint. The regulatory mechanism of cell cycle checkpoint is toensure cell cycle events occur in sequence and in a right way; it is also a key factor of the cell cycleregulation. Regulation of cell cycle checkpoint is done mainly through a series of enzymes. According toreports, the main regulatory enzymes involved in cell mitosis are: cell cycle of Plk1and Cdk1,cyclin-dependent protein kinase (cyclin-dependent kinases, CDKs), PP1, Cdc14phosphatase and spindlecheckpoint (spindle assembly checkpoint, SAC) and E3ubiquitin ligase anaphase-promoting complex andrelated molecules.Plk1(Polo-like kinase1) is an important serine-threonine protein kinase in cell mitosis, it plays anextremely important role in the process of cell mitosis. Plk1has different locations in the differentsub-phase of the cell cycle. Dozens substrate of Plk1have been disclosed so far, Plk1interacts with thesesubstrates to affect the cell cycle mitotic entry, spindle formation, sister chromatid separation, centrosomeduplication and cytoplasm splitting process; additionally, sub-cellular localization of Plk1is also affectedby the regulation of the corresponding substrate. In the occurrence and development process of humantumors, the most important manifestation is abnormal cell proliferation and increased migration capabilities.Plk1is a key molecule in the regulation of cell mitosis, Plk1expressed highly in tumor tissues is confirmedby a number of studies; in vitro cell culture and animal experiments, the low expression of Plk1caneffectively inhibit tumor cells proliferation and promote their own apoptosis, but it has no obvious effect onnormal cells. Therefore, in the treatment of human tumors, Plk1kinase targeted therapy is likely to become a potential new method.Erbin (ErbB2Interacting protein) is located in the junction of cell membrane and adjacent cell, itwas found and named in2000through yeast two-hybrid assay and the bait is cancer gene ErbB2. Ourlaboratory prepared Erbin antibody independently in early period, in order to detect Erbin antibody, we didimmuno-fluorescence assay and discovered that Erbin has a cell cycle-dependent expression increased andmitotic sub-cellular dynamic positioning characteristics, these findings prompted that Erbin in the cell cyclemay have an important role in the regulation of mitosis. We also found that Erbin has a mitosis-dependentphosphorylation character. Its phosphorylation is similar to Plk1activation and exist significant sub-cellularco-localization (centrosome and mesosome) in the cell cycle mitosis, suggesting that Erbin may be a newsubstrate protein of Plk1. Therefore, this topic intends to investigate the regulatory role and mechanism ofErbin as a new protein substrate of Plk1in cell mitosis.PurposesAt the cellular level to study whether the expression and phosphorylation of Erbin is cellcycle-dependent, Erbin sub-cellular localization changes of cell mitotic cycle, to reveal the impact of Plk1on Erbin phosphorylation and Plk1sub-cellular co-localization in mitosis, interactive domain of Erbin andPlk1. We also explored the impact of Erbin on cell mitosis preliminary.MethodsCell cycle synchronization experiments were used to observe the Erbin cell cycle-dependentexpression and its characteristics of phosphorylation, research the relationship between Erbin and Plk1phosphorylation in cell mitosis. Making use of immune fluorescence(IF) to detect dynamic changescharacteristics of Erbin sub-cellular location in cell cycles, co-localization of Erbin and Plk1in cell cyclemitosis, positioning foundation of interaction between endogenous/exogenous Erbin and Plk1, and to detectthe location of Erbin different truncated body thus to determine the basis of Erbin nuclear location.Molecular biology techniques were used to construct the corresponding vector. Co-immunoprecipitation(Co-IP) experiments confirmed initially the interaction of endogenous/exogenous Erbin with Plk1, makinguse of truncated body of Erbin different domain to further confirm Erbin and Plk1interactive domain, andGST-Pull down experiments were used to prove Erbin and Plk1interaction and their interactive domain. After sequence alignment analysis experiments, we speculated and confirmed Cdk1is the ignition kinase ofPlk1phosphorylated Erbin. si-Erbin experiments show that Erbin-knockdown interfered Plk1phospho-rylation; using flow cytometry and Nocodazole synchronization(M) release assay, after PI staining wedetected that si-Erbin interfered cell cycles. DAPI and Golgin-97staining nucleus and Golgi-body wereused to detect the interference on nucleus in mitosis after Erbin-knockdown, Erbin expression was knockeddown in normal human breast cells MCF-10A and we can observe the number of mitotic cells under aninverted microscope.ResultsFirst, we take advantage of the anti-microtubule drug Nocodazole to treat cells and make cellcycles arrested at the G2/M, analysis Erbin protein electrophoresis mobility of G2/M phase cell lysatesamples, and observed its migration slowed significantly, while λ-phosphatase treatment can flipNocodazole caused Erbin stripe migration slows, phosphorylation caused the shift of Erbin in the G2/Mphase, double thymidine was used to arrest cell at the G1/S phase and then release, observed Erbin stripswimming changes when cells naturally enter into the M phase, the results showed that Erbin stipe movesup significantly from G2phase to M phase (T9-T11), and accord with the pattern of kinase Plk1activation;Plk1knockdown inhibit Nocodazole caused the Erbin strip shift, expression of activated Plk1and wild-type Plk1can directly lead exogenous expression Erbin strip shift (transfect inactivated Plk1has no effect),the above experimental results confirmed the M phase Erbin phosphorylation depends on the kinase of Plk1.In addition, we have also preliminarily confirmed Cdk1is the ignition kinase of Plk1phosphorylated Erbin.We further make use of immunofluorescence to observe Erbin has significant mesosome and centrosomepositioning in cell cycles, Erbin and Plk1’s centrosome have significant co-localization in the early and midterm of mitosis, Erbin and Plk1’s mesosome have significant co-localization in the late term andcytokinesis. Co-immunoprecipitation experiments confirmed Erbin-PDZ domain interacts with Plk1’scatalytic region (Plk1-CD), the Erbin-Inter area interacts only with activated Plk1. And GST-Pull downexperiments are the further evidence of the interaction of Erbin and Plk1, Erbin-PDZ did not combine withPlk1-PBD, it combined with Plk1-CD, Plk1-PBD has a stronger interaction with phosphorylatedErbin-Inter district in vitro. These results suggest, Erbin is a new substrate of Plk1, substrate region ofErbin which is phosphorylated exists in the Erbin-Inter area, Erbin-PDZ may regulate Plk1activity. Our group has further confirmed that over-expression of PDZ can inhibit Plk1phosphorylation and delaymitotic entry. We also observed that cells division is significantly abnormal, mainly as multinucleated cellsenhanced through lentiviral infection knockdown Erbin15days later.ConclusionsThis study demonstrated Erbin has a cell cycle dependent expression and sub-cellular positioningdynamic characteristics of cell cycle mitosis, and also has a mitosis-dependent phosphorylationphenomenon, it exists significant interaction with Plk1kinase, and validates initially Cdk1is the "Ignition"kinase of Plk1-phosphorylated Erbin, substrate binding region of Plk1-phosphorylated Erbin exists inErbin-Inter area, Erbin-PDZ domain binds to Plk1-CD domain may interfere Plk1T210activation sterically.These results suggest that Erbin as the substrate of Plk1can regulate Plk1activity negatively; Erbin is animportant new regulatory molecules in cell mitosis. Erbin knockdown significantly promote cellproliferation and the generation of multinucleated cells, Erbin disorders mediated cell cycle abnormalitiesmay be closely associated with tumor development. Further revealed the molecular mechanism ofErbin-PDZ-mediated negative regulation of Plk1, may provide valuable information to explore new Plk1inhibition molecule drug.