Glycogen-Synthase-Kinase-3(GSK3) is a ubiquitously expressed, protein kinase found in all eukaryotes andexists in two isoforms; GSK3? and GSK3?. Evidence suggests that GSK3phosphorylates over 100 substrates(1,2,3), with the potential formany more still to be discovered. It acts as a downstream regulatory switch fornumerous signalling pathways, some of which include; cellular responses to WNT,insulin, G Protein Coupled Receptors (GPCR) and receptor tyrosine kinases(RTK). Consequently, GSK3 is involved in a huge variety of signal transductioncascades and so, influences many cellular functions such as cell proliferationand apoptosis, glucose regulation and cell cycle progression, just to name afew. Due to its involvement in these signalling pathways GSK3 has been linkedto multiple human chronic diseases including; Alzheimer’s Disease, Cancer andDiabetes.
(4) GSK3? has been linked to Alzheimer’sDisease pathobiology through its major role in the phosphorylation of tauprotein. Tau proteins act to stabilise microtubules however, when they becomehyperphosphorylated they consequentially fail to stabilise these microtubules,and this leads to the development of nervous system diseases such as Alzheimer’sDisease. In two studies by Forlenza, it was noted that patients withAlzheimer’s Disease had increased GSK3 activity(5) and that LithiumChloride may be used as a preventive treatment due to the inhibition of GSK3?.(6)The role of GSK3 in the Wnt signalling pathway has also been associated withcancer progression. However, GSK3 functions differently dependent on the tumourtissue type. In some tissue types GSK3 activity is diminished due tophosphorylation by growth factors and this has been linked to cancerprogression. Alternatively GSK3 can, as previously discussed, regulate the cellcycle and this is done via the Wnt/?-catenin signalling pathway.If this pathway were to become de-regulated, this has been linked to the developmentof breast cancer.
(7) In addition GSK3 has been known to negativelyregulate proto-oncogenes and therefore, can be said to have tumour suppressantfunctions.(8) In the mid-1990s GSK3 inhibition by insulin was foundto be crucial for insulin to enhance glycogen synthesis (glucose storage).Insulin signalling, as well as growth factors, inhibit GSK3 activity byphosphorylating Ser9 of GSK3 resulting in the blocked interaction between GSK3and pre-phosphorylated (primed) substrates.
Therefore, it could be consideredas a therapeutic target for diabetes. For example, if GSK3 is overexpressed or hyperactivated this would result in insulin resistance within type 2 diabetes. (4)Diseases such as these, which are all linked to GSK3, occur at a high frequencyworldwide and effect patients of all ages. There is a global need for new and moreeffective treatments to be discovered in order to combat these terrible,life-threatening and altering diseases. For these reasons GSK3 has beenproposed as a drug target, however, it is now known that GSK3 plays animportant role in fundamental cell physiology, hence the therapeutic window forinhibition of this kinase is very narrow.
Ideally, accurate biomarkers of GSK3activity are required in order to establish and quantify the relationshipbetween disease and GSK3 activity and subsequently, to titrate GSK3 inhibitorssafely and effectively in vivo.The5 cell lines that were investigated include; rat hepatoma H4, human fibroblastHEK293, mouse 3t3-L1, human neuroblastoma SHSY5Y and human lung cancer lineA549. The cell lines were treated with two GSK3 inhibitors; CT99021 (akaCHIR99021) and Lithium Chloride (LiCl). CT99021 is a cell permeable compoundacting as an ATP competitive and highly specific inhibitor of GSK3 activity.(9) Within animal models of disease, CT99021 has been found to increasethe power of insulin activation of glucose transport and utilisation, reducinginsulin resistance within muscle.
(10) Lithium Chloride is aselective inhibitor of GSK3 classically used as a mood stabiliser in theongoing treatment for Bipolar Disorder. In 1996 Klein and Melton (11)discovered that Lithium Chloride directly inhibited GSK3 activity as well asincreased the inhibitory-phosphorylation of GSK3 resulting in an amplificationof the direct inhibitory action.(12) Within muscle and adipocytecell lines Lithium Chloride has a stimulatory effect on glycogen synthesis andglucose transport. (10) Inorder to measure GSK3 activity in cells or in vivo one can directlyimmunoprecipitate GSK3 and assess the enzymatic activity towards a peptidesubstrate per unit of cellular protein. This is a useful measure to have but istechnically challenging and time consuming. In addition, it only provides asnapshot of the specific activity and represents the activity state of theisolated enzyme towards a peptide substrate. This may not be representative ofthe true enzyme activity present in multi-protein complexes, or of its activitytowards different substrates. Therefore an additional approach is to monitorthe phosphorylation of known GSK3 targets inside the cells or tissue ofinterest.
This provides a readout of GSK3 function, and includes the influenceof cellular location of GSK3 and phosphatases which would oppose any GSK3action. The main issue with the approach is the availability of sensitive andspecific antibodies to validated phosphorylation sites on GSK3 targets, alongwith the semi-quantitative nature of western blotting (which primarily allowscomparative studies rather than fully quantitative). To improve the approach,it would be beneficial to have a panel of validated antibodies to specific GSK3targets, a biomarker panel for GSK3 activity. My project aimed to investigatethe sensitivity and tissue selectivity of several antibodies to the proposedGSK3 targets detailed below. The ?-catenin/Wntpathway regulates cell fate decisions and stem cell pluripotency duringdevelopment Wnt signalling also acts as a negative regulator of GSK3. When theWnt-signal is absent, ?-catenin is targeted for sequential GSK3 phosphorylationby CK1 and the APC/Axin/GSK3?-complex. Within this complex, Axin acts as ascaffolding protein binding GSK3 and ?-catenin in close proximity, allowingfor the phosphorylation of ?-catenin. This leads to theubiquitination and proteasomal degradation of ?-catenin.
However, when Wnt ispresent it binds to Frizzled (Fz) receptors resulting in the activation ofDishevelled (Dsh). This processes leads to GSK3? being displaced from theAPC/Axin complex, thereby stabilising ?-catenin allowing transport to thenucleus and the regulation of target gene transcription. ?-catenin isassociated with various cancers due to its role as a proto-oncogene.
(13)By this process we would expect to see an increase in ?-Catenin due to adecrease in phosphorylation when GSK3 inhibitors are used. This has been shownin previous studies through the use of CT99021 (10) and LithiumChloride, which recovered Wnt signalling via the inhibition of GSK3?, resultingin ?-catenin stabilisation.(14) Therefore, changes in GSK3 activityshould associate with reciprocal changes in the total amount of ?-catenin inthe cell, hence total ?-catenin protein is a potential biomarker for GSK3activity. Collapsinresponse mediator protein 2 (CRMP2) is responsible for binding to microtubulesand regulating neuronal axon outgrowth.
This is a process regulated viasequential phosphorylation by GSK3 and cyclin-dependent kinase 5 (Cdk5), andoccurs specifically at sites that are hyperphosphorylated in Alzheimer Disease.It is possible that either GSK3 or Cdk5 overexpression, or a decrease in CRMP2phosphatase activity, would worsen the progression of Alzheimer’s disease, asthis would enhance CRMP2 and tau phosphorylation. In previous studies GSK3inhibitors, such as CT99021, caused rapid dephosphorylation of CRMP2 at Thr-509(15),and so this residue is a potential biomarker for GSK3 activity.
The enzyme Glycogen Synthase is a central regulator ofthe conversion of glucose into glycogen, contributing to the control of bloodglucose, by the dephosphorylation and activation of glycogen synthase. Glycogensynthase was once considered the best known substrate of GSK3 as it was one ofthe first to be discovered.(16) However, many more substrates havebeen discovered since its first report in scientific literature in 1982. Caseinkinase-2 (CK2) phosphorylates glycogen synthase as a requirement to form therecognition site for GSK3 phosphorylation, resulting in the reduction ofglycogen synthase activity.
Insulin inhibits GSK3 thereby relieving theinhibitory phosphorylation, promoting glycogen production and reducing bloodglucose. (9) This was the first GSK3 substrate reported andrepresents a validated substrate (at least in muscle). Therefore thephosphorylated glycogen synthase is a potential biomarker for GSK3 activity. Myc, a transcription factor, isinvolved in the stimulation of protein translation via the enhancement ofexpression of a variety of genes involved in the synthesis of nucleotides andribosomes.
(13) When GSK3 is overexpressed this results in enhancedphosphorylation, which facilitates the rapid proteolysis of C-Myc by theubiquitin pathway. In addition, GSK3 inhibition has been found to increaseC-Myc protein expression. (9) Therefore, total C-Myc protein is apotential biomarker for GSK3 activity. Finally,in addition to measuring these GSK3 targets my project aimed to assess totalGSK3 expression in each cell model (both GSK3 isoforms), along with assessingthe inhibitory phosphorylation of each GSK3 isoform (at Ser21 and Ser9 of GSK3?and GSK3?respectively).
In this way it would be possible to assess whether there were anydifferences in GSK3 isoform expression or regulation between the cell models.In addition I would assess whether any action of the GSK3 inhibitors on thebiomarkers included alterations in the amount, or regulation, of either GSK3isoform.