Studies Of AMPK's Non-metabolic Function:Substrate Identification And Roles In Cell Division

AMP-activated protein kinase(AMPK)is a highly conserved energy sensor composed of a catalytic a subunit,a scaffolding ? subunit,and a regulatory y subunit.In mammals,each subunit is encoded by multiple genes(?l,?2,?1 ?2,?1,?2,?3),generating at least 12 heterotrimeric combinations.AMPK is activated by stimuli such as nutrient deprivation,hypoxia and other cellular stresses that increase the AMP/ATP or ADP/ATP ratios,where in the binding of AMP or ADP to the ?subunit induces a conformational change that facilitates phosphorylation by upstream kinases while inhibiting dephosphorylation by protein phosphatases.Activated AMPK then promotes catabolic pathways to generate ATP while switching off ATP-consuming pathways thereby restoring energy homeostasis.Given its role as a central kinase in coordinating cell growth with energy status,the critical roles of AMPK in metabolic regulation have been widely studied,which also has made AMPK an attractive pharmacological target for treatment of metabolic diseases such as diabetes.However,in recent years the AMPK regulated pathways have been expanded to areas not directly viewed as metabolic processes,including cell metastasis,cell polarity and cell division.To fully understand the unexpected roles of AMPK in the context of expanding areas,in Chapter 2 of this thesis,we firstly performed a mass spectrometry(MS)-based quantitative phosphoproteomic study to identify direct AMPK phosphorylation sites.Combined with bioinformatic analysis,we screened out 20 new AMPK substrate candidates,8 of which were functionally associated with cytoskeleton and cell division.Then,in Chapter 3 we systemically investigated the roles of AMPK during cell division and found that AMPK is required for appropriate cell division progression.We also proved that KIF4A,a candidate of AMPK substrate identified in our phosphoproteomics study,is a direct AMPK substrate both in vitro and in vivo.We further elucidated how KIF4A-dependent central spindle length control is elaborately regulated by AMPK-and Aurora B-dependent phosphorylation in a competitive manner.As the AMPK-KIF4A regulation also responses to glucose stress,our work provides a new link between KIF4A-dependent central spindle length control and cellular energy stress.Microscopy based live cell tracking is a frequently used experiment method to study cellular processes like cell division.To date,the way to fulfill mitosis tracking of live cells is to express a fluorescence fused protein in advance,which therefore limits on cells easily been transfected or infected.Besides,the most widely used microtubule probes right now are based on taxane,which will greatly disturb the dynamic polymerization of microtubules resulting in an inability to track the normal process of cell division.In chapter 4,we show that graphene oxide greatly enhances the imaging ability of a peptide probe that selectively targets microtubules of the cytoskeleton,thus enabling the tracking of cell division in live cells...