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Novel application of KCTD12 protein in cell cycle control

A cell cycle, new application of technology, applied in the field of protein science

Active Publication Date: 2015-08-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific function of KCTD12 and its relationship with the cell cycle have not been reported so far.

Method used

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  • Novel application of KCTD12 protein in cell cycle control
  • Novel application of KCTD12 protein in cell cycle control
  • Novel application of KCTD12 protein in cell cycle control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Effect of KCTD12 on cell cycle in S phase

[0041] Synchronize the cells in the G1 / S phase, and add MG132 at a final concentration of 10 μM to treat the cells while releasing. MG132 is a proteasome inhibitor that inhibits proteasome degradation of ubiquitinated proteins. The cells of each phase were collected according to the method of synchronization, and the expression level of KCTD12 of the cells of each phase was detected by the method of Western Bolt ( figure 2 ). Then flow cytometry was used to detect the collected cells at each stage to detect the changes in their cycle ( image 3 ).

[0042] MG132 is a proteasome inhibitor that can inhibit the proteasome from degrading ubiquitinated proteins. figure 2 It indicated that MG132 significantly inhibited the degradation of KCTD12 in S phase and G2. image 3 It indicated that the addition of MG132 inhibited the degradation of protein KCTD12 and significantly blocked the process of cell cycle.

[0043]...

Embodiment 2

[0052] Example 2 Phosphorylation modification of KCTD12 under the regulation of Aurora kinase in M ​​phase

[0053] Since an important means of cell cycle regulation is to phosphorylate or dephosphorylate the protein to activate or inhibit the activity of the protein to achieve the purpose of regulation, it is speculated that KCTD12 may be phosphorylated, resulting in a larger molecular weight , the position moves up (in Western Bolt). In order to confirm the speculation, the protein in the M phase was treated with alkaline phosphatase (CIP), and the results Figure 4 It was shown that the position of KCTD12 shifted down after CIP treatment. It was confirmed that KCTD12 shifted up in M ​​phase because of phosphorylation modification.

[0054] After confirming that KCTD12 undergoes phosphorylation modification in the M phase, it is found that KCTD12 has an Aurora kinase action site by searching the database of phosphorylated proteins and comparing the motifs. From this, it w...

Embodiment 3

[0055] Example 3 Serine at position 243 is the phosphorylation site of KCTD12

[0056] The results of Example 2 confirm that KCTD12 is regulated by Aurora kinase in the M phase and undergoes phosphorylation modification. According to the pre-stored phosphorylation site, construct its mutation vector to determine its phosphorylation site. First construct the recombinant plasmid of pCMV-N-flag-KCTD12 on the backbone of pCMV-N-flag, and then perform point mutation on the basis of the already constructed pCMV-N-flag-KCTD12 to construct the phosphorylation site Mutation vector (mut1).

[0057] The plasmid pCMV-N-flag was used as the backbone to construct pCMV-N-flag-KCTD12. KCTD12 uses the cDNA of HeLa cells as a template, and designs primers for PCR amplification. The length of KCTD12 gene PCR product is about 1kb. Both the PCR product and the plasmid were digested with EcoR I and XbaI at 37°C for 4 hours, ligated overnight at 16°C, then transformed with E.coli competent, plate...

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Abstract

The present invention, belonging to the technical field of protein science, particularly discloses a new application of KCTD12 protein in cell cycle control. The KCTD12 protein cell control cycle is achieved via the following steps: regulating and controlling a cell cycle by controlling a degeneration degree of KCTD12 protein in an S period; or, regulating and controlling a cell cycle by controlling phosphorylation and dephosphorylation of KCTD12 protein in an M period; or, regulating and controlling a cell cycle by regulating interaction of the KCTD12 protein and CDK1 protein in an M period. The KCTD12 protein can serve as a new target of cancer cell proliferation regulation and control, and is favorable for developing more anti-cancer drugs.

Description

technical field [0001] The invention relates to the field of protein science and technology, in particular to the new application of KCTD12 protein in cell cycle regulation. Background technique [0002] The cell cycle refers to the process from the beginning of the end of cell division to the end of the next cell division and is the basis for the growth, development and function of multicellular organisms. The cell cycle of eukaryotic cells is divided into four phases, G1 phase, S phase, G2 phase and M phase, in which the S phase is responsible for DNA replication, and the M phase is responsible for the precise division of genetic material into two daughter cells. The S and M phases are more important than the other two in ensuring genetic stability. If errors occur in these two processes and are not corrected, it will have serious consequences, such as gene mutations, and may even lead to cancer. [0003] The highly ordered and precise life process of the cell cycle requ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12Q1/02A61K45/00A61K45/06A61P35/00
Inventor 何庆瑜钟颖
Owner JINAN UNIVERSITY
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