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    Home > Biochemistry News > Biotechnology News > Biochemical research in Rao Yi's laboratory: separation and purification of proteins revealing AMPK and its related kinases..

    Biochemical research in Rao Yi's laboratory: separation and purification of proteins revealing AMPK and its related kinases..

    • Last Update: 2022-05-11
    • Source: Internet
    • Author: User
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    In the past, Rao Yi's laboratory has long studied neurobiology and developmental biology with genetics and molecular biology


    Since 2011, the laboratory has concentrated a part of its energy and researchers to establish biochemical separation and purification methods in the laboratory


    The starting point is for neurobiological questions, but other biological questions may be involved


     


     

    The origins of these works lie in neurobiology


     

    Li Yang and Liu Yuxiang, graduate students in Rao Yi's lab, found that another site of SIK3 (T221) has an important function: its phosphorylation improves the function of SIK3 (Li Yang et al.


     

    SIK3 belongs to the AMPK family


     

    In 2003 and 2004, four labs published five articles arguing that the upstream kinase of AMPK was LKB1, which was previously found to be involved in suppressing a variety of tumors


     

    Liu Yuxiang and others from Rao Yi's laboratory found that LKB1 can indeed phosphorylate a specific site (T172) of AMPK, whether it is LKB1 expressed in mammals or LKB1 expressed in bacteria can phosphorylate T172 of AMPK


     

    Liu Ziyi of Rao Yi's lab made mutations in the gene that makes LKB1 in Drosophila and mice, and found that after the expression of LKB1 was reduced, sleep in both Drosophila and mice was reduced, indicating that LKB1 promotes sleep (Liu Ziyi et al.


     

    Using mammalian in vitro cultured cells (human HEK cells) and mouse brain, Liu Yuxiang found after column chromatography that in addition to LKB1, there were other activities of phosphorylating SIK3 and AMPK


     


     



     

    Liu Yuxiang (postgraduate/postdoctoral fellow), Wang Tao (graduate student), Cui Yunfeng (technician), Gao Shengxian (postdoctoral fellow), Rao Yi, etc.


     

    After the Rao Yi lab knocked out MST3 in mice, it was found that the mouse brain also had the activity of phosphorylating AMPK-T172 and SIK3-T221
    .
    After Liu Yuxiang knocked out MST3 in HEK cells, there were active phosphorylated AMPK-T172 and SIK3-T221 that were not LKB1
    .

     


    Liu Yuxiang performed protein separation and purification again and found CaMKK1 and 2
    .
    These two kinases were previously found to indeed phosphorylate AMPK-T172, but not as dramatically as LKB1
    .


    Liu Yuxiang found that, in addition to CaMKK, there is activity in HEK to phosphorylate AMPK-T172 and SIK3-T221
    .
    Liu Yuxiang et al.
    isolated and purified for the third time and found that MAP4K5 can phosphorylate AMPK-T172 and SIK3-T221
    .


    MST3 and MAP4K5, whether expressed in bacterial or mammalian cells, phosphorylate the 14 currently known AMPK-related kinases
    .
    Some of these have phosphorylated antibodies, and some have identified phosphorylation using protein mass spectrometry
    .

     

    MAP4K5 and MST belong to a subfamily of mammalian-like yeast sterile 20 protein kinases
    .
    The STE20 subfamily has 28 members
    .
    Liu Yuxiang, Wang Tao, Cui Yunfeng and others expressed all 28 protein kinases in bacteria and HEK
    .
    A dozen to two dozen of them were found to be able to phosphorylate AMPK and SIK3
    .
    The roles of these kinases on the two substrates AMPK and SIK3 are different, and the roles of different STE20 kinases are also different
    .



    Since 2016, Liu Yuxiang (postgraduate/postdoc), Wang Tao (postgraduate), Cui Yunfeng (technician), Li Chaoyi (postdoctoral), Jiang Lifen (postdoctoral), Rao Yi through in vitro biochemical prompts, a dozen or twenty STE20 protein kinases Phosphorylates AMPK and SIK3
    .

     

    The strength of these two articles is that through three rare rounds of biochemical separation and purification, it was found that the important protein kinase AMPK and its analogs can be phosphorylated by multiple members of the STE20 family
    .

     

    The weakness of these two articles is that the in vivo physiological implications of these in vitro biochemical findings are unclear
    .
    For example, it is unclear whether phosphorylation of AMPK by STE20 regulates metabolism
    .
    It is also unclear whether members of the STE20 family can regulate sleep
    .

     

    If the STE20-AMPK upstream and downstream relationships are indeed involved in many and important physiological processes, these findings are important breakthrough discoveries
    .

     

    If the STE20-AMPK upstream and downstream relationships are not involved in important physiological processes in vivo, these findings are beautiful biochemistry in vitro, but similar to the moon in water, they are of little use other than viewing
    .

     

     

    Liu YX, Wang TV, Cui YF, Gao SX and Rao Y (2022).
    Biochemical purification uncovers mammalian sterile 3 (MST3) as a new protein kinase for multifunctional protein kinases AMPK and SIK3.
    J Biol Chem 2022.
    101929.

     

    Liu YX, Wang TV, Cui YF, Li CY, Jiang LF and Rao Y (2022).
    STE20 phosphorylation of AMPK related kinases revealed by biochemical purifications combined with genetics.
    J Biol Chem 2022.
    101928.

     


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